CN104278046A - Hepatitis B virus and chlamydia trachomatis chimeric nucleic acid vaccine and application thereof - Google Patents

Abstract

The invention relates to a hepatitis B virus and chlamydia trachomatis chimeric nucleic acid vaccine and application thereof. More concretely, the invention relates to a chimeric nucleic acid sequence containing a nucleic acid sequence encoding chlamydia trachomatis main-outer-membrane-protein multi-epitope protein and a nucleic acid sequence encoding hepatitis B virus surface antigen, a recombinant vector and a host cell both containing the chimeric nucleic acid sequence, a preparation method for the recombinant vector, and application of the host cell to prepare the chimeric vaccine for preventing or treating hepatitis B virus and chlamydia trachomatis correlated diseases. The vaccine has efficient safe immunoprophylaxis and treatment effects on HBV and Ct correlated diseases, the preparation technology and the immunization process are simple, the effect is obvious and the repeatability is strong.

Description

Hepatitis B virus and chlamydia trachomatis chimeric nucleic acid vaccine and application thereof

technical field

The present invention relates to the fields of biomedical technology and immunology, in particular to a chimeric nucleic acid vaccine of hepatitis B virus and Chlamydia trachomatis, its preparation method, and its role in preventing and treating hepatitis B virus infection and related tumors (such as primary liver cancer) and trachoma Application in Chlamydia infection.

Background technique

Hepatitis B virus (HBV) infection is a global public health problem, and my country is a high prevalence area of hepatitis B. Chronic HBV infection threatens human health, persistent infection can develop into chronic hepatitis, liver cirrhosis or liver cancer. Currently there is no effective treatment for hepatitis B, and hepatitis B vaccination is an effective way to prevent and control hepatitis B.

Chlamydia trachomatis (Ct) is one of the main pathogens of sexually transmitted diseases (STDs). Cause male urethra and female cervicitis after infection, it is reported that 50-60% of male urethra is caused by Ct infection (Tiodorovi.J, Randelovi.G, Koci.B et al, Bacteriological finding in the urethra in men with and without non-gonococcal urethritis, 2007, 64(12): 833-6), and often mixed with other pathogens, infecting the female urogenital tract through sexual transmission, leading to adverse pregnancy, such as miscarriage, stillbirth, etc., and is closely related to cervical cancer relevant. Ct infection is very easy to relapse after treatment. WHO estimates that there are about 90 million Ct patients in the world, and it costs billions of dollars to treat Ct infection every year (Wang SA, Papp JR, Stamm WE et al, Evaluation of antimicrobial resistance and treatment failures for Chlamydia trachomatis: a meeting report. J Infect Dis. 2005, 191(6): 917-23.).

In recent years, the incidence of STDs caused by Ct in my country has been on the rise, which has caused great harm to human health. For HBV and Ct, especially for Ct infectious diseases, there is still no effective prevention and treatment method so far. It is one of the important topics for workers in related disciplines in various countries to seek effective vaccines, especially the research on vaccines that can prevent and treat HBV and Ct infectious diseases at the same time.

Nucleic acid vaccine, that is, DNA vaccine, is a new type of vaccine, which consists of a gene encoding the target antigen and a plasmid expression vector. After being directly introduced into the host cell, it does not integrate with the host chromosome, but expresses the protein antigen through the transcription system of the cell. Induce the body to produce specific cellular immunity and humoral immunity.

The development of nucleic acid vaccines is called the "third vaccine revolution" after complete pathogen vaccines and genetically engineered recombinant protein vaccines. Compared with traditional attenuated or inactivated vaccines, nucleic acid vaccines have the advantages of being convenient for storage and transportation; compared with subunit vaccines, nucleic acid vaccines have the ability to activate the body's overall immune response (cellular immune response and humoral immune response) for a long time, prepare The advantages of simple process and low cost (no need to purify protein).

Studies have shown that: hepatitis B virus surface antigen (HBsAg) has good immunogenicity and has a good effect on preventing hepatitis B virus infection. It has become a protein vaccine for preventing hepatitis B virus infection and has been widely used. HBsAg is a protein with sugar groups connected by disulfide bonds, on which some exogenous antigens can be inserted to form a chimeric protein to enhance the immunogenicity of exogenous proteins (Michel ML, Manciai M, RiviereY, et al. al. T and B lymphocyto responses to Human Immunodeficiency Virus (HIV) Type l in Macaques immunized with hybrid HlV/hepatitis Bsurface antigen particles. J of virology, 1990; 64(5): 2452-2455). At present, research on HBsAg DNA vaccine has shown that it has the same immune protection effect as HBsAg protein (generating specific humoral immunity and cellular immunity).

Ct has 19 serotypes, of which serotypes such as D-H mainly cause urogenital tract infections, and types E and D are reported at home and abroad (see: Chen Lili, Wu Yimou, Deng Zhongliang, etc. Urogenital infections in some cities in southern China Genotyping of Chlamydia trachomatis. Chinese Journal of Dermatology, 2006, 39: 275-277; Suchland RJ, Eckert LO, Hawes SE et al, Longitudinal assessment of infecting serovars of Chlamydia trachomatis in Seattle, public healthclinics: 1988-1996. The most common type in Sex Transm Dis, 2003, 30:357-361.). Ct mainly uses its main outer membrane protein (MOMP) as the basis of vaccine research (see: Pal S, Theodor I, Peterson EM, de la MazaLM., Immunization with the Chlamydia trachomatis mouse pneumonitis major outer membrane protein can elicit a protective immune response against a genital challenge. Infect Immun, 2001, 69(10): 6240-7; and Zhang D, Yang X, Berry J et al. DNA vaccination with the majorouter-membrane protein gene induces acquired immunity to Chlamydia trachomatis (mousepneumonitis) infection . Infect Dis. 1997;176(4):1035-40).

Current Ct vaccine research includes: ① subunit vaccine: MOMP or the peptide chain of its variable region is used as the antigen, but the effect is not satisfactory. The main reason may be that the antigenicity of MOMP is different for each serotype. ② Recombinant vaccine: MOMP gene is cloned into Escherichia coli through genetic engineering, so that it can be expressed efficiently, and it can be used as a vaccine after separation and purification. Its immune protection effect needs to be further studied. ③Synthetic peptide vaccine: Based on the variable region of MOMP, select CTL or Th epitopes, and artificially synthesize peptide chains, which can produce certain protective cellular and humoral immunity, but the immunogenicity is weak. ④ DNA vaccine: the gene encoding MOMP or a certain segment of the peptide chain on it is cloned into a plasmid and used as a vaccine. Since the production of the antigen is endogenous and has a natural structure, it can induce the body to produce cellular immunity and humoral immunity. The most promising Ct vaccine in current research.

Since the natural MOMP protein contains disulfide bonds, it is difficult to prepare and maintain its activity, and studies have shown that it may cause pathological immune responses, so multiple T cell epitopes (including CTL epitopes and CTL epitopes and Th epitope) and B cell epitope, composed of Ct MOMP multi-epitope (Ct MOMP multiepitope, Ct MOMPm), the multi-epitope gene was cloned into eukaryotic plasmid and identified to immunize mice, respectively in mouse serum and vagina Specific antibodies IgG, sIgA and specific CTL cells against Ct were detected in the secretion, and they had a certain protective effect on the challenge of Ct in the reproductive tract of the mouse model. However, the immunogenicity of this epitope DNA vaccine is weak, so further research is needed to enhance its immunogenicity and immune protection (see: Zhu Shanli, Shi Chaohui, Li Wenshu, etc. Immunogenicity of the main outer membrane protein multi-epitope gene of Chlamydia trachomatis Research. Chinese Journal of Preventive Medicine, 2009, 43(3): 232-236).

In summary, there is an urgent need in this field to develop a vaccine that can enhance the immunogenicity and immunoprotective effect of Ct multi-epitope DNA vaccines, and at the same time have a multivalent vaccine that can prevent and treat multiple diseases, especially a vaccine that can simultaneously prevent and treat Safer and highly effective nucleic acid vaccines for HBV and Ct.

Contents of the invention

The object of the present invention is to provide a chimeric nucleic acid vaccine of hepatitis B virus and chlamydia trachomatis which can be used for simultaneously preventing and treating hepatitis B virus infection, its related tumors (such as primary liver cancer) and chlamydia trachomatis infection.

In a first aspect of the present invention, a chimeric nucleic acid sequence is provided, said sequence comprising:

(a) a nucleic acid sequence encoding the major outer membrane protein polyepitope protein of Chlamydia trachomatis; and

(b) Nucleic acid sequence encoding hepatitis B virus surface antigen.

In a preferred example, the polyepitope protein of the main outer membrane protein of Chlamydia trachomatis is an amino acid sequence containing multiple CTL epitopes, Th epitopes and B cell epitopes.

In another preferred example, the amino acid sequences of the plurality of CTL epitopes, Th epitopes and B cell epitopes are arranged in series.

In another preferred example, the nucleic acid sequences in (a) and (b) are modified by human codon optimization.

In another preferred example, the nucleic acid sequence encoding the polyepitope protein of the major outer membrane protein of Chlamydia trachomatis is connected to the carboxy-terminal or amino-terminal of the nucleic acid sequence encoding the surface antigen of hepatitis B virus.

In another preferred example, the nucleic acid sequences in (a) and (b) are connected directly or through a connecting sequence.

In one embodiment of the present invention, the sequence of the polyepitope protein of the main outer membrane protein of Chlamydia trachomatis is shown in SEQ ID NO:2.

In another embodiment of the present invention, the sequence of the hepatitis B virus surface antigen is shown in SEQ ID NO:5.

In another embodiment of the present invention, the nucleic acid sequence of the main outer membrane protein multi-epitope protein of the coding Chlamydia trachomatis is selected from: SEQ ID NO: 1 (i.e. the original sequence without humanized codon optimization) and SEQ ID NO: 1 ID NO: 3 (i.e. the humanized codon-optimized sequence).

In another embodiment of the present invention, the nucleic acid sequence encoding the surface antigen of hepatitis B virus is selected from the group consisting of: SEQ ID NO: 4 (i.e. the original sequence without humanized codon optimization) and SEQ ID NO: 6 (i.e. humanized codon-optimized sequences).

In the second aspect of the present invention, a recombinant vector is provided, said vector comprising the above-mentioned chimeric nucleic acid sequence of the present invention.

In a preferred example, the vector is selected from: eukaryotic expression vectors or prokaryotic expression vectors, preferably bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, or mammalian cell viruses, more preferably pcDNA3.1 (+), pSIREN -NEO, pET32a(+), pQE30, pGEX-4T-1, pPICZA.

In the third aspect of the present invention, a genetically engineered host cell containing the above-mentioned vector of the present invention is provided.

In a preferred example, the host cell is selected from prokaryotic cells, lower eukaryotic cells or higher eukaryotic cells, preferably animal cells, Escherichia coli, or yeast, more preferably COS-7 cells, COS-1 cells, E .coli or GS115.

In the fourth aspect of the present invention, the use of the above-mentioned chimeric nucleic acid sequence, recombinant vector or host cell of the present invention in the preparation of chimeric vaccines for preventing or treating diseases related to hepatitis B virus and Chlamydia trachomatis infection is provided.

In a preferred example, the diseases related to hepatitis B virus and Chlamydia trachomatis are selected from: hepatitis B virus infection, tumors associated with hepatitis B virus infection, or Chlamydia trachomatis infection, preferably hepatitis B, primary liver cancer, urethritis, cervicitis, trachoma .

In the fifth aspect of the present invention, a composition is provided, which comprises an effective amount of the aforementioned chimeric nucleic acid sequence, recombinant vector or host cell of the present invention and pharmaceutically or immunologically acceptable carriers and excipients or adjuvants.

In a preferred example, the composition is an immune composition or a pharmaceutical composition, preferably a vaccine.

In a preferred example, the carrier, excipient or adjuvant is selected from: liposome, starch, gelatin, aluminum salt adjuvant, saponin adjuvant or Ribi adjuvant.

In another preferred example, the composition contains 0.01-99.9wt% of the recombinant vector of claim 4, preferably 0.1-99.0wt%.

In another preferred embodiment, the composition is intramuscular injection, skin immunization or mucosal immunization.

In a sixth aspect of the present invention, there is provided a method for preparing the above-mentioned recombinant vector of the present invention, the method comprising:

(i) Provide the nucleic acid sequence encoding the polyepitope protein of the main outer membrane protein of Chlamydia trachomatis and the nucleic acid sequence encoding the surface antigen of hepatitis B virus;

(ii) connecting said nucleic acid sequences to form a chimeric nucleic acid sequence;

(iii) Cloning the chimeric nucleic acid sequence obtained in step (ii) into a vector.

In a preferred example, the sequence of the main outer membrane protein polyepitope protein of Chlamydia trachomatis is shown in SEQ ID NO: 2. The sequence of the hepatitis B virus surface antigen protein is shown in SEQ ID NO:5.

In another preferred example, the nucleic acid sequence encoding the main outer membrane protein polyepitope protein of Chlamydia trachomatis is selected from the group consisting of: SEQ ID NO: 1 (i.e. the original sequence without humanized codon optimization), SEQ ID NO: 3 (i.e. humanized codon-optimized sequences).

In another preferred example, the nucleic acid sequence encoding the surface antigen of hepatitis B virus is selected from the group consisting of: SEQ ID NO: 4 (i.e. sequence without humanized codon optimization), SEQ ID NO: 6 (humanized codon-optimized sequence) sub-optimized sequence).

In another preferred example, the nucleic acid sequence encoding the polyepitope protein of the major outer membrane protein of Chlamydia trachomatis is connected to the carboxy-terminal or amino-terminal of the nucleic acid sequence encoding the surface antigen of hepatitis B virus.

It should be understood that other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Description of drawings

Figure 1: Identification map of pcDNA3.1(+)/Ct MOMPm-HBsAg recombinant plasmid.

In the figure, "M1" means: DL1000 DNA marker; "1" means: recombinant plasmid pcDNA3.1(+)/CtMOMPm-HBsAg; "2" means: pcDNA3.1(+)/Ct MOMPm-HBsAg/EcoR I+Xho I; "3" means: Ct MOMPm PCR product; "4" means: pcDNA3.1(+)/Ct MOMPm-HBsAg/HindIII+EcoR I; "5" means: HBsAg PCR product; "M2" means: DL250 DNA marker .

Figure 2: Identification map of pcDNA3.1(+)/HBsAg-Ct MOMPm recombinant plasmid.

In the figure, "M1" means: DL1000 DNA marker; "1" means: recombinant plasmid pcDNA3.1(+)/HBsAg-CtMOMPm; "2" means: pcDNA3.1(+)/HBsAg-Ct MOMPm/EcoR I+Xho I; "3" means: HBsAg PCR product; "4" means: pcDNA3.1(+)/HBsAg-Ct MOMPm/BamH I+EcoR I; "5" means: Ct MOMPmPCR product; "M2" means: DL250DNA marker .

Figure 3: Detection of HBsAg expression in COS-7 cells by indirect immunofluorescence.

Figure 4: Indirect immunofluorescence detection of Ct MOMPm expression in COS-7 cells.

Figure 5: RT-PCR identification of the mRNA transcribed from the HBsAg target gene in the eukaryotic cells of the chimeric recombinant plasmid.

In the figure, "M" means: 100bp DNA Ladder; "1" means: RT-PCR product of COS-7 cells transfected with pcDNA3.1(+)/Ct MOMPm plasmid; "2" means: pcDNA3.1(+) /Ct MOMPm-HBsAg plasmid transfected COS-7 cell RT-PCR product; "3" means: pcDNA3.1(+)/HBsAg-Ct MOMPm plasmid transfected COS-7 cell RT-PCR product; "4" Indicates: RT-PCR product of COS-7 cells transfected with pcDNA3.1(+)/HBsAg plasmid; "5" indicates: RT-PCR product of COS-7 cells transfected with pcDNA3.1(+) plasmid.

Figure 6: RT-PCR identification of the mRNA transcribed by the Ct MOMPm target gene in the eukaryotic cells of the chimeric recombinant plasmid.

In the figure, "M" means: 100bp DNA Ladder; "1" means: RT-PCR product of COS-7 cells transfected with pcDNA3.1(+)/Ct MOMPm plasmid; "2" means: pcDNA3.1(+) /Ct MOMPm-HBsAg plasmid transfected COS-7 cell RT-PCR product; "3" means: pcDNA3.1(+)/HBsAg-Ct MOMPm plasmid transfected COS-7 cell RT-PCR product; "4" Indicates: RT-PCR product of COS-7 cells transfected with pcDNA3.1(+)/HBsAg plasmid; "5" indicates: RT-PCR product of COS-7 cells transfected with pcDNA3.1(+) plasmid.

Figure 7: Dynamic changes of Ct-specific IgG antibodies in serum of mice in each group after immunization.

Figure 8: Dynamic changes of HBsAg-specific IgG antibodies in serum of mice in each group after immunization.

Figure 9: Dynamic changes of Ct-specific sIgA antibody in vaginal secretions of mice in each group after immunization.

Figure 10: Specific CTL killing rate of splenic lymphocytes of immunized mice on target cells carrying Ct MOMP polyepitope protein antigen.

Figure 11: Vulva inflammation of mice in each group after Ct challenge.

Figure 12: Observation of pathological sections of the reproductive system of mice in each group after Ct challenge.

Figure 13: Isolation and culture of reproductive tract Ct and IFU counting of mice in each group after Ct challenge.

Detailed ways

The present inventors have prepared HBsAg-Ct MOMP multi-epitope chimeric DNA vaccine based on the HBsAg and Ct MOMP multi-epitope genes and optimized the humanized codons. Immunization experiments have proved that the HBsAg-Ct MOMP multi-epitope chimeric DNA vaccine can both prevent and treat Ct infection in the reproductive tract of mice, and its immune control effect on Ct is significantly better than that of non-chimeric Ct MOMP multi-epitope vaccine. On this basis, the inventors have completed the present invention.

Specifically, the inventors analyzed and predicted the immunodominant regions of multiple serotypes of Ct MOMP through biological software, screened shared amino acid sequences containing multiple CTL epitopes, Th epitopes and B cell epitopes, and analyzed the The gene was modified by human codon optimization, and the Ct MOMP multi-epitope gene was obtained. At the same time, the inventors also modified the human codons in the HBsAg gene. Then, the HBsAg-Ct MOMP multi-epitope chimeric DNA vaccine was prepared by molecular biology techniques.

The inventors further tested the immune protective effect of the DNA vaccine by immunizing mice: ELISA was used to detect the serum IgG and vaginal secretion sIgA antibody levels of mice after immunization, and the CTL-specific killing activity of spleen lymphocytes to evaluate the epitope vaccine. Immune effect and immune protection in mouse genital tract infection Ct model. The results showed that the HBsAg-CtMOMPm DNA vaccine had both immune prevention and treatment effects on Ct infection in the reproductive tract of mice, and its immune control effect on Ct was significantly better than that of the non-chimeric Ct MOMP multi-epitope vaccine.

The present invention proves through experimental research that the prepared HBsAg-Ct MOMP multi-epitope chimeric DNA vaccine can stimulate the body to produce stronger specific humoral immune effects against HBsAg and Ct MOMP, especially protective antibodies to local genital tract mucosa, And the cellular immunity against Ct MOMP has the effect of preventing and treating HBsAg and Ct infection at the same time, laying the foundation for the in-depth research and application of using one vaccine to prevent and treat multiple diseases, and has important scientific research value.

At the same time, the present invention also verifies the HBsAg-Ct MOMP multi-epitope chimeric gene through animal experiments, and enhances the immune effect of the Ct MOMP multi-epitope gene. The results of the study confirmed that the HBsAg-Ct MOMP multi-epitope chimeric gene can induce the body to produce enhanced Ct-specific humoral immunity and cellular immunity, especially the local sIgA antibody response in the mucosa, and these immune effects are significantly better than non-chimeric Ct MOMP multi-epitope vaccine, thereby enhancing the immune protection effect.

Glossary

The term "immunological activity" or "immunogenicity" herein refers to the ability of a natural, recombinant or synthetic vaccine to induce a specific humoral and/or cellular immune response in a mammal. As used herein, the terms "nucleic acid vaccine", "DNA vaccine" or "chimeric (nucleic acid) vaccine" refer to a chimeric nucleic acid sequence of the invention that elicits an immune response in a mammal.

The term "immune response" herein includes cellular and/or humoral immune responses sufficient to suppress or prevent infection; or to prevent or suppress diseases caused by hepatitis B virus and Chlamydia trachomatis.

As used herein, the term "subject" or "individual" or "patient" refers to any subject in need of diagnosis or treatment, especially a mammalian subject, especially a human, other subjects including cattle, dogs, cats, guinea pigs, rabbits, rats , mice, horses, etc. Of particular concern are those subjects who are susceptible or have been infected with HBV and/or C. trachomatis.

The terms "nucleic acid" and "nucleic acid sequence" herein refer to nucleotides (ribonucleotides or deoxyribonucleotides) of any length in polymeric form. It includes (but is not limited to) single- and double-stranded DNA or RNA, genomic DNA and cDNA.

The term "effective amount" or "immunologically effective amount" herein refers to an amount administered to an individual as a single dose or a fraction of consecutive doses that is effective for treatment or prophylaxis. The amount depends on the health and physiological status of the individual to be treated, the category of the individual to be treated (such as non-human primates, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection required, the preparation of the vaccine, and the treating physician's opinion on medical treatment. Depends on the assessment of the situation, and other relevant factors. This amount is expected to lie within a relatively wide range and can be determined by routine experimentation.

chimeric nucleic acid sequence

The chimeric nucleic acid sequence of the present invention comprises: (a) nucleic acid sequence encoding Chlamydia trachomatis major outer membrane protein multi-epitope protein (Ct MOMP); and (b) nucleic acid sequence encoding hepatitis B virus surface antigen.

The term "Chlamydia trachomatis major outer membrane protein multi-epitope protein (Ct MOMP)" refers to an amino acid sequence containing multiple MOMP CTL epitopes, Th epitopes and B cell epitopes. The multi-epitope protein can be obtained by predicting and screening the common epitope genes of multiple serotype Ct MOMPs, and will simultaneously contain multiple HLA-A2 specific CTL epitopes and Th and B cell epitopes to form a multi-table bit gene. For example, the network resource database can be used to obtain the MOMP gene and amino acid sequence of each serotype of Ct, and the HLA-A*0201-restricted T cell epitope (CTL), Th epitope and B cell For epitope prediction, the dominant epitopes with high scores are selected and connected into multi-epitope genes, preferably in series. The dominant epitopes include (but are not limited to): HLA-A2 restricted CTL epitopes (1-9aa, 18-26aa, 16-24aa, 26-34aa, 27-35aa, 29-37aa, 38-46aa, 40-48aa), Th epitopes (8-27aa) and B cell epitopes (2-15aa), including h-2d type CTL epitopes (18-26aa).

The term "hepatitis B virus surface antigen (HBsAg)" refers to the main structural protein of hepatitis B virus - hepatitis B surface antigen, its coding sequence is known in the art (see for example Yong-Lin Y, Qiang F, Ming-Shun Z, et al. Hepatitis B surface antigen variants in voluntary blood donors in Nanjing, China. Virol J. 2012Apr14; 9(1): 82. doi: 10.1186/1743-422X-9-82.). Those of ordinary skill in the art should understand that these known HBsAg coding sequences can be used in the chimeric nucleic acid sequences of the present invention.

It should be understood that the nucleic acid sequence of the present invention may also include molecules that hybridize to the above-mentioned coding sequence under stringent conditions and have the same or similar activity. The term "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60° C.; or (2) denaturing agent added during hybridization, Such as 50% (v/v) formamide, 0.1% calf serum/O.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 50%, preferably more than 55% , 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more or 90% or more, more preferably 95% or more, hybridization occurs. For example, the sequence may be a complementary sequence.

In a preferred embodiment of the present invention, the Ct MOMP coding sequence or the HBsAg coding sequence of the present invention is modified with human codon optimization, so that the resulting gene can obtain higher expression in the expression system. Preferably, one or both of the chimeric nucleic acid sequences are nucleic acid sequences modified by human codon optimization. The "human codon optimization modification" can be carried out using conventional methods known in the art (for example, Vaccine candidate MSP-1 from Plasmodium falciparum: a redesigned4917bp polynucleotide enablesynthesis and isolation of Pan W, RavotE, Tolle R, etc. full-length protein from Escherichia coli and mammalian cells. Nucleic Acids Res. 1999 Feb5 15; 27(4): 1094-103). Preferably, the GC content in the nucleic acid sequence is increased by 5-40%, preferably 10-30%, more preferably 13-23%, but the encoded amino acid remains unchanged.

The nucleic acid sequences of the present invention may be linked directly, or via a linker sequence. The term "connecting sequence" herein refers to a sequence that is located between the nucleic acid sequence encoding the polyepitope protein of the main outer membrane protein of Chlamydia trachomatis and the nucleic acid sequence encoding the surface antigen of hepatitis B virus. The length of the linker sequence is not particularly limited, usually 0-100. The length of the connecting peptide can also be 0, which means that the gene sequences are directly connected. Typically, the linking sequence has no or no significant effect on the expression of the linked sequence and the immune effect of the antigen produced.

In a preferred embodiment of the present invention, the Ct MOMP coding sequence is connected to the carboxy-terminal or amino-terminal of the HBsAg coding sequence.

Vectors and host cells

After obtaining the chimeric nucleic acid sequence encoding the present invention, it can be connected into a suitable expression vector, and then transformed into a suitable host cell.

In the present invention, the term "vector" and "recombinant vector" are used interchangeably, and refer to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. In the present invention, the carrier selected from the following group can be used: eukaryotic expression vector or prokaryotic expression vector, preferably bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, or mammalian cell virus, more preferably pcDNA3.1 (+), pSIREN - NEO, pET32a, pQE30, pGEX-4T-1 or pPICZA.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell or an animal cell. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells and the like. In the present invention, COS-7 cells, COS-1 cells, E. coli, and GS115 are preferably used.

The nucleic acid vaccine of the present invention is composed of a gene encoding the target antigen and an expression vector of a plasmid, which is not integrated with the host chromosome after being directly introduced into the host cell, but expresses the protein antigen through the transcription system of the cell, and induces the body to produce specific cellular immunity and humoral immunity.

combination

The present invention also provides various compositions comprising the recombinant vector of the present invention, including pharmaceutical compositions and vaccine compositions. The composition can be used to prevent or treat diseases related to hepatitis B virus and chlamydia trachomatis, said diseases include (but not limited to): hepatitis B virus infection, tumors associated with hepatitis B virus infection, or chlamydia trachomatis infection, preferably hepatitis B, primary liver cancer , Urethritis, cervicitis, trachoma.

Various compositions comprising the recombinant vector of the present invention may contain a buffer selected according to the actual use of the recombinant vector; and may also contain other substances suitable for the intended use. The choice of buffering agent is well within the skill of the art, and a variety of buffering agents are known in the art to be suitable for the intended use. In some instances, the composition may contain a pharmaceutically acceptable excipient, many of which are known in the art and need not be discussed in detail here. Various pharmaceutically acceptable excipients are described in various publications, including, for example, "Remington's Pharmaceutical Sciences" ("Remington's Pharmaceutical Sciences", 19th Edition (1995) Mack Publishing Co.).

The pharmaceutical composition or vaccine composition can be prepared into various dosage forms, such as injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, suppositories, transdermal drugs (such as patches, etc.), ointments, Lotion etc. Pharmaceutical grade organic or inorganic carriers and/or diluents, suitable for oral or topical use, may be used in formulating various compositions containing the therapeutically active compounds. Diluents known in the art include aqueous media, vegetable and animal oils and fats. Stabilizers, wetting agents and emulsifiers, salts to change the osmotic pressure or various buffers and skin penetration enhancers to maintain a suitable pH value can also be used as auxiliary materials.

When used as a vaccine, the recombinant vector of the present invention can be formulated in various ways. Generally, the vaccine or medicine of the present invention is formulated with suitable pharmaceutical carriers and/or vehicles according to various methods well known in the art. A suitable carrier is sterile saline. Other aqueous and nonaqueous isotonic sterile injection solutions and aqueous and nonaqueous sterile suspensions (all known pharmaceutically acceptable carriers well known to those skilled in the art) may also be used for this purpose.

In addition, the preparation of the vaccine composition of the present invention may also contain other components, including, for example, adjuvants, stabilizers, pH regulators, preservatives and the like. These components are well known to those skilled in the vaccine art. Adjuvants include (but are not limited to) aluminum salt adjuvants; saponin adjuvants; Ribi adjuvants (Ribi ImmunoChem Research In., Hamilton, MT); Montanide ISA adjuvants (Seppic, Paris, France); Hunter's TiterMax Adjuvant (CytRx Corp., Norcross, GA); Gerbu adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Germany) and the like. In addition, other components that modulate the immune response (IL-12, CpG oligodeoxynucleotides (CpG-ODN), etc.) may also be included in the formulation.

Route of Administration and Dosage

When used as a vaccine, the recombinant vector of the present invention can be administered to a subject by a known method. These vaccines are usually administered by the same route of administration as conventional vaccines and/or by simulating pathogen infection routes. When in the form of a vaccine composition, a pharmaceutically acceptable carrier may also be included. In addition, this composition may also include adjuvants, flavoring agents or stabilizers and the like.

Conventional and pharmaceutically acceptable routes of administration of the pharmaceutical or vaccine compositions of the present invention include: intranasal, intramuscular, intratracheal, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral or other parenteral Route of administration. The routes of administration can be combined if desired, or adjusted according to the antigenic peptide or disease state. Vaccine compositions may be administered in single or multiple doses, and may include administration of booster doses to elicit and/or maintain immunity.

The recombinant vaccine should be administered in an "effective amount", that is, the amount of the recombinant vector is sufficient to trigger an immune response in the selected route of administration, and can effectively promote the protection of the host against hepatitis B virus and Chlamydia trachomatis infection or the symptoms of hepatitis B and Chlamydia infection.

The amount of recombinant vector used in each vaccine dose is determined according to the amount that can elicit an immune protective response without obvious side effects. Typically, each dose of vaccine is sufficient to produce about 1 μg-10 mg, preferably 5 μg-2 mg, more preferably 10 μg-1 mg of protein after infection of host cells. The effective dose of the vaccine calculated based on the recombinant vector nucleic acid usually includes administration of about 1 μg-10 mg chimeric nucleic acid/kg body weight, preferably 1 μg-10 mg chimeric nucleic acid/kg body weight, more preferably 1 μg-10 mg chimeric nucleic acid/kg body weight. The optimal amount for a particular vaccine can be determined by standard research methods, including observation of antibody titers and other responses in subjects. Monitor the level of immunity provided by the vaccine to determine if a booster dose is needed. After assessment of antibody titers in sera, a booster dose of immunization may be required. Administration of adjuvants and/or immunostimulants can enhance the immune response to the proteins of the invention.

Main advantages of the invention

The main advantages of the present invention are:

(1) Provide a kind of HBsAg-Ct MOMP multi-epitope chimeric DNA vaccine, described chimeric DNA vaccine can induce body to produce effective HBsAg and Ct MOMP specific humoral immunity and cellular immunity, relevant to HBV and Ct simultaneously The disease has immune prevention and treatment effect;

(2) Compared with Ct MOMP multi-epitope DNA vaccine, the vaccine of the present invention has an effective immune effect on Ct, and can be used for more effective prevention and treatment of Ct-related diseases;

(3) The preparation process of the vaccine of the present invention is simple, low in cost, convenient and safe to use, easy to store and transport, and can activate the overall immune response of the body for a long time, and has the advantages of simple immunization process, safe inoculation, obvious effect, and strong repeatability.

Example

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to routine conditions (for example, can refer to people such as Sambrook, "Molecular Cloning Laboratory Guide" (New York: Cold Spring Harbor Laboratory Press, 1989) or Immunology Methods Manual, Ivan Lefkovits, CRC, 1998) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Embodiment 1. Modification and acquisition of HBsAg gene

Using the serum samples of HBsAg-positive patients in Wenzhou area as a template, PCR primers were designed and synthesized, and restriction sites (BamH I and EcoR I) were introduced at both ends to amplify the complete open reading frame of HBsAg, and the PCR product was combined with the vector pcDNA3.1( +) were ligated after double digestion at the same time to construct the recombinant plasmid pcDNA3.1(+)/HBsAg(wt), which was identified by sequencing.

In order to enhance the expression of HBsAg, the full-length HBsAg nucleotide (SEQ ID NO: 4) was modified and designed, that is, human-preferred codons were optimized, and glycosylation sites and transcription termination sequences were excluded. The 3rd position is a synonymous codon for G and C, ensuring that the optimized amino acid is completely consistent with the wild type, increasing its GC content from 42.4% to 60.1%, but keeping the encoded amino acid unchanged (SEQ ID NO: 5) . Add BamH I and EcoR I restriction sites to both ends of the optimized and modified HBsAg base sequence, and entrust Shanghai Sangon Bioengineering Company to synthesize the whole gene sequence. The synthesized HBsAg codon-optimized sequence was digested with BamH I and EcoR I and connected to the vector pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1(+)/HBsAg(optimized), which was identified by sequencing.

Example 2. Preparation and in vitro expression analysis of HBsAg-Ct MOMP multi-epitope DNA vaccine

Design and synthesize PCR primers, introduce restriction sites EcoR I and Xho I at both ends, and amplify the PCR product with the human codon-optimized Ct MOMP multi-epitope gene as a template, and pass the PCR product through EcoR I and Xho After I digested, it was connected to the recombinant plasmid pcDNA3.1 (+)/HBsAg (optimized), and the recombinant eukaryotic expression plasmid containing the Ct MOMP multi-epitope gene at the carboxy-terminal of HBsAg was constructed, and was digested by restriction enzymes (Fig. 1, 2) and Sequencing identification. The results showed that the chimeric recombinant plasmids of HBsAg-Ct MOMPm and Ct MOMPm-HBsAg were constructed successfully.

The resulting recombinant eukaryotic plasmids were transfected into COS-7 cells, and HBsAg and Ct MOMPm specific primers were used respectively to detect the mRNA transcription of HBsAg and Ct MOMPm genes in COS-7 cells after transfection by RT-PCR (Fig. 3, 4). The results showed that the HBsAg of about 700bp and the Ct MOMPm gene DNA fragment of 170bp were obtained respectively, which were consistent with the expected target gene fragment size (Fig. Ct MOMPm-specific primers were used for PCR amplification, and none of the obvious target bands were amplified, indicating that the above RT-PCR products were derived from the transcribed mRNA after the recombinant plasmid was transfected into COS-7 cells.

The expression of the recombinant plasmid in COS-7 cells was further identified by indirect immunofluorescence (Fig. 5, 6). The results showed that: using human HBsAb-positive serum as the primary antibody, pcDNA3.1(+)/HBsAg, pcDNA3.1(+)/HBsAg-Ct MOMPm, and pcDNA3.1(+)/CtMOMPm-HBsAg were all expressed in COS-7 cells. Can express HBsAg protein (green fluorescent dots in the cytoplasm). The serum of rabbits immunized with the whole body of Chlamydia trachomatis was used as the primary antibody. -7 cells can express Ct MOMP multi-epitope protein (green fluorescent spots in the cytoplasm).

The results indicated that the recombinant plasmid containing HBsAg-Ct MOMPm could be efficiently expressed in eukaryotic cells in vitro.

Example 3. The immune protection effect of HBsAg-Ct MOMP multi-epitope DNA vaccine

Female BALB/c mice aged 6-8 weeks (purchased from Shanghai Slack Experimental Animal Co., Ltd.) were selected and randomly divided into 6 groups, 21 mice in each group, respectively: PBS control group, pcDNA3.1(+) group , pcDNA3.1(+)/HBsAg group, pcDNA3.1(+)/HBsAg-Ct MOMPm recombinant plasmid group, pcDNA3.1(+)/Ct MOMPm-HBsAg recombinant plasmid group and Ct MOMPmDNA immune group. 100 μl of test samples (1 μg/μl) were taken at 0, 2, and 4 weeks respectively, and the mice were injected into the quadriceps femoris muscle for immunization. Tail vein blood and vaginal secretions were collected at Ow, 2w, 4w, 6w, 8w, 10w, 12w, 14w, 18w, and 22w after immunization, and ELISA method was used to detect IgG antibodies in serum and sIgA antibodies in secretions; The spleen cell suspension was prepared from the spleen, and the specific killing activity of CTL against Ct MOMP was detected by lactate dehydrogenase (LDH) release method. The remaining mice in each group were challenged with E serotype Ct, and the activities of the mice and the severity of vulvar inflammation were observed every day after the challenge, and photographed and recorded.

(1) Determination results of serum specific antibody IgG of immunized mice:

Using the indirect ELISA method, the purified Ct whole cells and HBsAg were coated on a polyethylene micro-reaction plate, and after sealing, 1:50 diluted serum of immunized mice was added, reacted at 37°C for 2 hours, washed the plate, and added 1:2000 dilution respectively. 100 μl of HRP-goat anti-mouse IgG, reacted at 37°C for 2 hours, after washing the plate, o-phenylenediamine (OPD) developed color, added 2 mol/L H ₂ SO ₄ to terminate the reaction, and measured the A490 value with a microplate reader. Each serum sample was tested repeatedly in 3 wells, and the serum specific antibody IgG level of immunized mice in each group was determined. The results are shown in Figures 7 and 8.

The results showed that the IgG level of Ct-specific antibody in the serum of mice in the HBsAg-Ct MOMPm immunization group, Ct MOMPm-HBsAg immunization group, and Ct MOMPm immunization group increased with the number of immunizations, and the corresponding antibody level continued to increase. It was the highest, then gradually decreased, and there was still a certain antibody level at 18w (Figure 7); the antibody levels of the Ct MOMPm-HBsAg group and the Ct MOMPm group were close, and they were lower than the HBsAg-Ct MOMPm group at 8-14w (F= 216.96, P<0.05), but it was significantly higher than that in the pcDNA3.1(+) group and the PBS control group at 2 to 18w, and the difference was statistically significant (F=153.04, P<0.05).

At the same time, the mice after immunization could also produce IgG antibodies specific to HBsAg ( FIG. 8 ). HBsAg-specific antibody IgG levels in HBsAg-Ct MOMPm group, Ct MOMPm-HBsAg group, and HBsAg group mouse serum increased with the increase in the number of immunizations, and the antibody level continued to increase. It reached a peak at 1 w after the last immunization, and remained at a certain level until 22 w. Compared with the pcDNA3.1(+) group and the PBS group, there were significant differences (F=234.31, P<0.05).

(2) Determination of specific antibody sIgA in secretions of immunized mice:

The indirect ELISA method was adopted, and the method was the same as above, and the results were shown in FIG. 9 .

The results showed that the level of specific antibody sIgA in the vaginal secretions of HBsAg-Ct MOMPm group, Ct MOMP m-HBsAg group, and Ct MOMPm group increased with the number of immunizations, and the corresponding antibody level continued to increase. It reached the peak 2w after the last immunization, and then gradually decreased, but the HBsAg-Ct MOMPm group decreased more slowly. HBsAg-Ct MOMPm group mouse vaginal secretion specific antibody sIgA level compared with Ct MOMPm-HBsAg group, Ct MOMPm group, pcDNA3.1(+) group and PBS, there were significant differences at 4-10w (F =196.67, P<0.05); at the same time, the level of specific antibody sIgA in the vaginal secretions of mice in the Ct MOMPm-HBsAg group was also higher than that in the Ct MOMPm group, and the difference was also statistically significant (F=17.4, P<0.05).

(3) Results of specific CTL killing activity of immunized mice:

The spleen was taken to prepare a splenocyte suspension to adjust the concentration to 2×10 ⁶ cells/ml, and the specific killing activity of CTL was detected by lactate dehydrogenase (LDH) release method. The results are shown in Figure 10.

After immunization, when the ratio of effector cells to target cells (E:T) of HBsAg-Ct MOMPm immunized mice was 20:1, 10:1 and 5:1, the specific CTL killing activity against Ct MOMPm was higher than that of Ct MOMPm group, Ct MOMPm-HBsAg group, and the killing activity of other control groups all have significant difference (P＜0.05) (Fig. 10);

(4) Immunoprotective effect of HBsAg-Ct MOMPm DNA vaccine on mouse genital tract Ct infection:

Two weeks after the last immunization of mice in 6 groups including PBS, pcDNA3.1(+), pcDNA3.1(+)/HBsAg group, HBsAg-Ct MOMPm group, and Ct MOMPm-HBsAg group, the E serotype Chlamydia trachomatis standard strain ( purchased from the American Type Collection (ATCC: VR-348B)) for reproductive tract infection.

The results showed that from the first day after transvaginal Ct infection, the mice in each group began to show signs of infection to varying degrees, such as redness and swelling of the vulva and abnormal secretions. The mice in the HBsAg-Ct MOMPm group showed more severe inflammation at the beginning, but the inflammation subsided faster than the other groups, and lasted only about 15 days; About 5 days shorter than control mice such as PBS (Fig. 11).

The results of pathological changes in the reproductive system of mice after Ct challenge in reproductive tract (Figure 12): PBS group, pcDNA3.1(+) group, HBsAg group mice vaginal Ct challenge caused different degrees of reproductive tract infection and lesions: vaginal edema; oviduct Hydrops, swelling, lumen thinning, swelling of epithelial cells, and disappearance of folds; the scope of inflammation spread to the ovary, causing ovarian inflammatory cell infiltration and congestion; while the degree of inflammation in the vagina, fallopian tubes, and ovaries in the HBsAg-Ct MOMPm and Ct MOMPm immunized groups were all the same. The results were relatively mild, which indicated that HBsAg-Ct MOMPm recombinant plasmid and Ct MOMPm recombinant plasmid DNA immunization had a certain inhibitory effect on the damage, delay and spread of Chlamydia trachomatis infection.

The results of Ct IFU detected by the mouse vaginal secretion cell culture method after Ct challenge showed that (Fig. 13), after 10 days of Ct challenge, the amount of Ct IFU detected by mice in the HBsAg-Ct MOMPm group was lower than that of mice in other groups (F ＝81.77, P＜0.05); while Ct MOMPm-HBsAg group and Ct MOMPm group had close detection amount of Ct IFU in vaginal secretion, and after 10 days of Ct challenge, they were higher than HBsAg-Ct MOMPm plasmid group but lower than control group (pcDNA3.1(+)/HBsAg, pcDNA3.1(+), PBS); Among them, the detection amount of Ct IFU in the vaginal secretions of mice in the Ct MOMPm plasmid group was second only to the HBsAg-Ct MOMPm group.

in conclusion:

A series of animal experiments were carried out using the prepared HBsAg-Ct MOMP multi-epitope DNA vaccine, and the results confirmed that:

1. The experimental animals produced CtMOMP-specific protective antibodies against the HBsAg-Ct MOMP multi-epitope DNA vaccine in serum and vaginal secretions. The vaccine can induce Ct-specific CTL killing activity after immunization, indicating that it can produce a strong anti-Ct cellular immune effect. The results show that it has the function of preventing and treating Ct infection.

2. Experimental animals produced HBsAg-specific antibodies in serum against HBsAg-Ct MOMP multi-epitope DNA vaccine. The results show that it has the effect of preventing HBV infection.

3. Experimental animals produced significantly enhanced serum IgG, secreted sIgA and specific CTL killing activity against HBsAg-Ct MOMP multi-epitope DNA vaccine (especially compared with Ct MOMP multi-epitope DNA vaccine). The results showed that the immune effect of Ct MOMP multi-epitope DNA could be enhanced by preparing a vaccine chimerized with HBsAg.

4. Mice inoculated with HBsAg-Ct MOMP multi-epitope DNA vaccine have strong immunopreventive and therapeutic effects on E-type Ct infection. It shows that the HBsAg-Ct MOMP multi-epitope DNA vaccine immunization has a good protective effect on Ct reproductive tract challenge, and the effect of the vaccine has been exerted.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (8)

1. a chimeric nucleic acid sequence, described sequence is connected to nucleotide sequence (b) by nucleotide sequence (a) and is formed, wherein:

(a) coding Major Outer Membrane Protein of Chla mydia trachomatis multi-epitope albumen and through people's source pin optimize modify nucleotide sequence; With

(b) coding hepatitis B virus surface antigen and through people's source pin optimize modify nucleotide sequence, described nucleotide sequence is as shown in SEQ ID NO:6.

2. chimeric nucleic acid sequence as claimed in claim 1, it is characterized in that, the aminoacid sequence of described hepatitis B virus surface antigen is as shown in SEQ ID NO:5.

3. chimeric nucleic acid sequence as claimed in claim 1, it is characterized in that, the nucleotide sequence of described coding hepatitis B virus surface antigen is selected from: SEQ ID NO:4 and SEQ ID NO:6.

4. a recombinant vectors, described carrier comprises chimeric nucleic acid sequence according to claim 1.

5. a genetically engineered host cell, described host cell contains carrier according to claim 4.

6. chimeric nucleic acid sequence, recombinant vectors according to claim 4 or host cell according to claim 5 as claimed in claim 1 purposes in the Chimeric DNA vaccine for the preparation of prevention or treatment hepatitis B virus and chlamydia trachomatis relative disease.

7. a composition, it includes recombinant vectors according to claim 4 and acceptable carrier, vehicle or the adjuvant pharmaceutically or in immunology of effective amount, and wherein said composition is Chimeric DNA vaccine.

8. prepare a method for recombinant vectors described in claim 4, described method comprises:

I () provides coding Major Outer Membrane Protein of Chla mydia trachomatis multi-epitope albumen and optimizes through people's source pin the nucleotide sequence modified, and coding hepatitis B virus surface antigen is provided and optimizes through people's source pin the nucleotide sequence modified, described nucleotide sequence is as shown in SEQ ID NO:6;

(ii) connect described nucleotide sequence, form chimeric nucleic acid sequence;

(iii) step (ii) gained chimeric nucleic acid sequence is cloned in carrier.