CN1305898C - Stimulated polypeptide, its coding genes and use of capsule protein HVR1 of hepatitis C virus - Google Patents

Abstract

The invention discloses eight hepatitis C virus envelope protein hypervariable region 1 (hypervariable region 1, HVR1) mimic polypeptides and their coding genes. They are capable of extensive cross-reactivity with patient sera. At the same time, the present invention also provides their uses.

Description

Hepatitis C virus envelope protein HVR1 mimic polypeptide, its coding gene and application

technical field

The invention relates to the field of genetic engineering, in particular to hepatitis C virus envelope protein HVR1 mimic polypeptide with extensive cross-reactivity, its encoding gene and its application.

Background technique

Hepatitis C virus (HCV) is the main pathogen that causes post-transfusion hepatitis and sporadic non-A non-B hepatitis. %-80% of HCV infection leads to viral persistence and chronic liver disease, and 20%-30% of them can progress to liver cirrhosis or even hepatocellular carcinoma.

HCV is a single-stranded positive-sense RNA virus with an envelope, belonging to the Hepacivirus genus of the Flaviviridae family. The genome is about 9500 bases and is divided into a 5' untranslated region, an open reading frame (ORF) and a 3' untranslated region. . ORF encodes a polyprotein precursor of about 3000 amino acids, which is processed into 10 viral proteins under the joint action of host cell signal enzymes and viral proteases, which are Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B.

Hypervariable region 1 (hypervariable region 1, HVR1) is located at the N-terminal of E2, nt1150-1230, aa384-410, immediately downstream of the signal peptide sequence (aa370-383), about 27aa. HVR1 is the most variable segment of the entire virus genome, and it has great differences among virus strains, individuals, and even different periods of the same individual, and is the main reason for the heterogeneity of infected viruses between individuals and within individuals. Studies have shown that HVR1 is an important target of neutralizing antibodies in the host's humoral immunity, but its strain specificity can cause some viruses to evade the host's immune recognition and persist in infection. Nevertheless, there are relatively conserved sites in the HVR1 sequence, and it is possible to induce protective antibodies through appropriate HVR1 immunization procedures (Farci P, Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region l of the envelope E2 protein .Proc Natl Acad SciUSA 1996，96：15394-15399；Esumi M，Experimental vaccine activities ofrecombinant E1 and E2 glycoproteins and hypervariable region 1peptides of hepatitis C virus in chimpanzees.Arch Virol1999，144：973-980；Cerino A，Monoclonal antibodies with broad specificity for hepatitis C virus hypervariable region 1 variants can recognize viral particles. J Immunol. 2001, 167: 3878-3886).

There have been many studies using HVR1 natural sequences or mimic peptide sequences to find highly cross-reactive HVR1 sequences to obtain candidate immunogens as specific immune prevention and treatment of hepatitis C (Puntoriero G, Towards a solution for hepatitis C virus hypervariability : mimotopes of the hypervariable region l can induce antibodies cross-reacting with a large number of viral variants. EMBO J.1998, 17: 3521-3533). The commonly used method is to synthesize HVR1 peptides, but the cost is expensive and the variation types are limited. Using DNA shuffling or molecular breeding (Figure 1) (Stemmer WPC.. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci USA. 1994, 91: 10747-10751; Minshull J, Stemmer WPC.. Protein evolution by molecular breeding. Current Opinion in Chem biol 1999, 3: 284-290.), has not been reported yet.

Currently there is no effective HCV vaccine, and the treatment methods for HCV are quite limited. It is very important to develop effective HCV vaccine and therapeutic drugs.

Therefore, there is an urgent need in the art to develop mimetic polypeptides with HCV immunogenicity and their coding sequences.

Contents of the invention

The purpose of the present invention is to provide a kind of mimetic polypeptide with HCV immunogenicity and extensive cross-reactivity with patient serum and its coding sequence.

Another object of the present invention is to provide the application of such mimetic polypeptide and its coding sequence.

In the first aspect of the present invention, a class of hepatitis C virus envelope protein HVR1 mimic polypeptide is provided, characterized in that, the mimic polypeptide has SEQ NO: 2, 4, 6, 8, 10, 12, 14 or 16 shown amino acid sequence.

In the second aspect of the present invention, the fusion protein of the simulated polypeptide is provided, characterized in that, the fusion protein includes any polypeptide selected from SEQ NO: 2, 4, 6, 8, 10, 12, 14, 16 Two or more amino acid sequences. Preferably, the fusion protein includes the amino acid sequences shown in SEQ NO: 2 and SEQ NO: 4.

In another preferred example, the fusion protein includes any three or four amino acid sequences selected from SEQ NO: 2, 4, 6, 8, 10, 12, 14, and 16.

In the third aspect of the present invention, a class of hepatitis C virus envelope protein HVR1 mimic gene is provided, characterized in that the mimic gene encodes the mimic polypeptide of the present invention.

In a preferred example, the mimic gene has the nucleotide sequence shown in SEQ NO: 1, 3, 5, 7, 9, 11, 13 or 15.

In the fourth aspect of the present invention, there is provided the use of the mimetic polypeptide, which is characterized in that the mimetic polypeptide is used in the preparation of drugs for detecting, preventing and/or treating hepatitis C.

In a preferred example, the mimic polypeptide is used to prepare a vaccine for preventing and/or treating hepatitis C.

In the fifth aspect of the present invention, the application of the fusion protein is provided, which is characterized in that the fusion protein is used in the preparation of drugs for detecting, treating and/or preventing hepatitis C.

In a preferred example, the fusion protein is used to prepare a vaccine for preventing and/or treating hepatitis C.

In the sixth aspect of the present invention, the application of the mimic gene is provided, which is characterized in that the mimic gene is used in the preparation of drugs for detecting, treating and/or preventing hepatitis C.

In the seventh aspect of the present invention, a vector is provided, which is characterized in that it contains the mimetic gene of the present invention.

In the eighth aspect of the present invention, a genetically engineered host cell is provided, characterized in that it is a host cell transformed or transduced by the vector of the present invention.

In the ninth aspect of the present invention, there is provided a method for preparing a hepatitis C virus envelope protein HVR1 mimic polypeptide, characterized in that the method comprises:

(a) cultivating the host cell of the present invention under conditions suitable for expressing the hepatitis C virus envelope protein HVR1 mimic polypeptide;

(b) Isolation of the hepatitis C virus envelope protein HVR1 mimetic polypeptide from the culture.

In the tenth aspect of the present invention, an antibody capable of specifically binding to the mimetic polypeptide of the present invention is provided, including monoclonal antibodies and polyclonal antibodies.

In the eleventh aspect of the present invention, a detection kit is provided, which is characterized in that it contains the mimetic polypeptide of the present invention, the antibody of the present invention, the fusion protein of the present invention or a combination thereof. Preferably, the detection kit contains the mimetic polypeptide of the present invention.

In the twelfth aspect of the present invention, there is provided a method for in vitro detection of hepatitis C virus antigen or anti-HCV antibody in a sample, characterized in that it comprises the steps of:

(a) Contacting the sample with a substance selected from the group consisting of the mimetic polypeptide of the present invention, the antibody of the present invention, the fusion protein of the present invention or a combination thereof.

(b) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of HCV antigen or anti-HCV antibody in the sample.

In a preferred example, the sample is serum.

In another preferred example, the substance is the mimetic polypeptide of the present invention.

In another preferred embodiment, the substance is the fusion protein of the present invention.

In another preferred example, the detection method in the step (b) is ELISA or fluorescence detection.

The polypeptide of the present invention refers to "hepatitis C virus envelope protein HVR1 mimic polypeptide" or "hepatitis C virus envelope protein HVR1 mimic gene product".

Polypeptides of the invention may be the product of chemical synthesis, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, phage, insect and mammalian cells).

In the present invention, the term "hepatitis C virus envelope protein HVR1 mimic polypeptide" refers to a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16. The term also includes variants of the above amino acid sequences that have the same function as the hepatitis C virus envelope protein HVR1 mimic polypeptide. These variations include (but are not limited to): deletions, insertions and/or substitutions of several (usually 1-5) amino acids, and addition of one or several (usually 15) amino acids at the C-terminal and/or N-terminal Within, preferably within 10, more preferably within 5) amino acids. For example, adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein. For example, in the present invention, the envelope protein containing the mimic polypeptide sequence is obtained by replacing the original HVR1 sequence of the envelope protein with the mimic polypeptide sequence.

The coding region sequence encoding the polypeptide of the present invention may be the same as the coding region sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to encoding a protein having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, but not identical to SEQ ID NO: Nucleic acid sequences that differ from the coding sequences shown in 1, 3, 5, 7, 9, 11, 13 or 15.

The term "polynucleotide encoding a polypeptide of the present invention" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The nucleotide sequence of the hepatitis C virus envelope protein HVR1 mimic gene can usually be obtained by PCR amplification, recombination or artificial synthesis.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

At present, the DNA sequence encoding the polypeptide of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations may also be introduced into the polypeptide sequences of the invention by chemical synthesis.

The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering with the vector of the present invention or the coding sequence of the hepatitis C virus envelope protein HVR1 mimic polypeptide, and the production of the polynucleotide described in the present invention through recombinant technology. Peptide method.

By conventional recombinant DNA technology (Science, 1984; 224:1431), the polynucleotide sequence of the present invention can be used to express or produce a recombinant hepatitis C virus envelope protein HVR1 mimic polypeptide. Generally speaking, there are the following steps:

(1). Use the polynucleotide (or variant) encoding the hepatitis C virus envelope protein HVR1 mimic polypeptide of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolating and purifying the hepatitis C virus envelope protein HVR1 mimic polypeptide from culture medium or cells.

Specifically, the hepatitis C virus envelope protein HVR1 mimic polypeptide of the present invention can be introduced into the host cell by introducing the corresponding coding sequence (directly or by introducing a vector containing the hepatitis C virus envelope protein HVR1 mimic polypeptide coding sequence), and in The transformed host cells are cultivated under appropriate conditions to express the hepatitis C virus envelope protein HVR1 mimic polypeptide, and then the hepatitis C virus envelope protein HVR1 mimic polypeptide is isolated and purified.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; or chimeric antibodies, such as with murine antibodies Antibodies that bind specificity but retain the antibody portion from humans.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified hepatitis C virus envelope protein HVR1 mimic gene product can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing the hepatitis C virus envelope protein HVR1 mimetic polypeptide can be used to immunize animals to produce antibodies.

The monoclonal antibodies of the present invention can be produced using hybridoma technology. All kinds of antibodies of the present invention can be obtained through conventional immunization techniques by using the hepatitis C virus envelope protein HVR1 to mimic gene products. These simulated gene products can be produced by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies that bind to unmodified forms of the hepatitis C virus envelope protein HVR1 mimic gene products can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (such as sugar sylated or phosphorylated polypeptides), which can be obtained by immunizing animals with gene products produced in eukaryotic cells (eg, yeast or insect cells).

The production of polyclonal antibodies can be used to immunize animals, such as rabbits, sheep, etc., with the hepatitis C virus envelope protein HVR1 mimic polypeptide. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

The hepatitis C virus envelope protein HVR1 mimic polypeptide or the fusion protein and the mimic gene can be used to prepare medicines for detecting, preventing and/or treating hepatitis C. In particular for the preparation of vaccines for the prevention and/or treatment of hepatitis C.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the hepatitis C virus envelope protein HVR1 mimic polypeptide of the present invention, the fusion protein or its antisense nucleic acid, and a pharmaceutically acceptable carrier or excipient . Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 0.1 microgram/kg body weight to about 1.0 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

A method for detecting whether there is a specific antibody to the hepatitis C virus envelope protein HVR1 in a sample is to use the hepatitis C virus envelope protein HVR1 analog polypeptide to detect, which includes: contacting the sample with the hepatitis C virus envelope protein HVR1 analog polypeptide ; Observe whether the antibody complex is formed, and the formation of the antibody complex means that there is hepatitis C virus in the sample.

The polynucleotide of the hepatitis C virus envelope protein HVR1 mimic polypeptide can be used for the diagnosis and treatment of diseases related to the hepatitis C virus envelope protein HVR1.

The inventors can rapidly change the gene sequence by using DNA shuffling or molecular breeding technology, and its most notable progress is the in vitro transformation of natural diversity, that is, the simulation as close as possible to the natural sequence. The basic principle is that a group of related genes are randomly cut into small fragments, and the homologous sequences on each fragment serve as primers for each other during annealing, and are reconnected by mutual recombination to form new sequences. Figure 1 is a schematic diagram of the basic principle of the technology.

The present invention adopts the method of DNA shuffling to recombine in vitro the natural sequence of 31 strains of HVR1 cDNA obtained from the serum of hepatitis C patients, constructs a phage display library of HVR1 analog sequence, and obtains eight HVR1 with extensive cross-reactivity through affinity screening of anti-HVR1 polyclonal antibody By simulating the polypeptide and its coding gene, its reactivity is increased from 46.7%-66.7% to 53.3%-80%. The two most reactive HVR1 mimetic peptides are SEQ NO: 2 (80%) and SEQ NO: 4 (76.7%), suggesting that they may be important immunogenic peptides, and their sequences are highly homologous to the consensus sequence. The reason for its broad reactivity. On this basis, two or more sequences can be expressed in series (fusion expression) to form a multi-epitope antigen (fusion protein) to further increase the reaction rate. At the same time, prepare antibodies with corresponding synthetic peptides or expression products, observe the ability of antibodies to neutralize (capture) viruses, and lay the foundation for further inducing corresponding antibodies with broad reactivity in the body.

Description of drawings

Figure 1. Schematic diagram of the basic principle of DNA shuffling (molecular passage technology)

Figure 2. Agarose gel electrophoresis results of PCR amplification products of the original HVR1 fragment.

1 is the PCR Marker; 2 is the fragment amplified by PCR without primers after DNase I digestion; 3 is the fragment amplified by PCR with specific primers; 4 is the negative control; 5-11 are the original HVR1 amplified from the pGEMT-E plasmid fragment.

Detailed ways

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 methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Example 1: Preparation of mock HVR1 fragments by DNA shuffling

In this study, primers HCp7 and HCp8 were used to PCR amplify 31 HVR1 cDNA sequences from the recombinant pGEMT-E plasmid (the eDNA fragment of the HCV envelope region amplified from the serum of patients with hepatitis C was inserted into the pGEM-T vector (Promega)) After purification, it was digested with DNase I, and 10 μl of the digested product was electrophoresed on a 2% agarose gel. Mock HVR1 fragment. The experimental results are shown in Figure 2. In Figure 2, 1 is the PCRMarker; 2 is the fragment amplified by PCR without primers after DNase I digestion; 3 is the fragment amplified by PCR with specific primers; 4 is the negative control; 5-11 are the fragments amplified from the pGEMT-E plasmid Raw HVR1 fragment.

HCp7: 5'ATAC GGCCCAGCCGGCC ATGGCCGCCGTTGACGGG 3'

Sfi I

HCp8: 5' CATTGAAT GCGGCCGC GTTTACAAGCTG 3'

Not I

Example 2: Construction of HVR1 Mimetic Peptide Phage Display Library

The recovered HVR1 fragment was cloned into the pCANTAB 5E vector (Pharmacia) between the Sfi I and Not I sites, making it fused to the 5' end of the main coat protein (pIII) gene of M13 phage, and the ligated product was desalted and purified Afterwards, the competent Escherichia coli XL1-blue (Stratagene Company) was electrotransformed, and the original library size was 1.96×10 ⁶ . In order to verify the quality and complexity of the HVR1 library, we randomly selected 10 clones for sequencing identification. The results showed that the inserted HVR1 sequences of the 10 clones were all different, so the constructed library was considered to be of high complexity.

After the phage library was amplified and purified by precipitation with polyethylene glycol (PEG8000), the pellet was resuspended in 5ml TBSB. Take 10 μl for dilution and titrate, the concentration is 1.46×10 ¹² cfu/ml.

Example 3: Biological Screening of HVR1 Mimetic Peptide Library

As mentioned above, the binding rates of the four DHFR-HVR1 fusion proteins (SH1b, BJ1b, SD1b, SD2a) expressed in Escherichia coli to the anti-HCV positive hepatitis C patient serum were 66.7% (20/30), 60% (18/30), 53.3% (16/30) and 46.7% (14/30). Four HCV-infected sera that could react with the four HVR1 fusion proteins were selected, and affinity screening was performed on the HVR1 library in turn. In order to further enhance the reactivity of the mimic peptides with different HVR1 antibodies, we used different sera to conduct a second round of affinity screening on the enriched phages, and healthy blood donor serum was used as a control. The enrichment rate increased from 10 in the first round. ^-7 to 10 ^-3 in the fourth round, an improvement of 10 ⁴ times (Table 1).

Round of panning phage applied phage eluted enrichment factor 1234 8.8×10 ¹⁰ 5.3×10 ¹⁰ 2.9×10 ¹⁰ 2.5×10 ¹⁰ 3.0×10 ⁴ 1.3×10 ⁶ 3.9×10 ⁶ 4.0×10 ⁷ 3.4×10 ^-7 2.4×10 ^-5 1.3×10 ^-4 1.6×10 ^-3

Table 1 Biological screening results of HVR1 phage display library

Example 4: Identification of HVR1 Mimetic Peptides

After four rounds of biological screening, 8 different phage clones were selected and amplified respectively. The expressed eight mimetic peptides can be widely recognized by the HVR1 antibody in the blood of HCV-infected patients. The binding rate of the purified phage clones to anti-HCV positive sera was detected by ELISA method, and the results showed that the reaction rates of the 8 clones and 30 sera were 53.3%-80.0% (Table 2). Among them, clone No. 1 had the highest reactivity. The determination of the DNA sequence of the inserted mimetic peptide showed that none of the selected clones encoded the same HVR1 peptide as the original virus strain.

The reaction rates of 30 sera with 8 HVR1 mimic peptides (see Table 3 for sequences) were different, 10 sera could react with all 8 phage clones, 12 sera could react with 5-7 clones, There were 4 sera that reacted with 1-4 clones and 4 sera that did not react with any phage clone. The specific experimental results are shown in Table 2.

Table 2 shows the reactivity of 8 strains of HVR1 mimetic peptides with 30 sera of hepatitis C patients. The peptidomimetic name is annotated at the top of each column. The number of each serum is indicated on the left side of each row. Shown in the table is the ratio of each specimen to the negative mean OD value. The bottom end of each column indicates the respective reaction rates of 8 mimetic peptides with serum and their total reaction rates.

Table 2: HCV HVR1 mimetic peptide and serum reactivity test results of patients with hepatitis C serial number amino acid sequence 1 ETYVSGGSAARNAYGLTSLFTVGPAQK 2 ETYVTGGVAGRTTLGFTSLFTPGPPSQ 3 ETYVTGGMAGRTTLGFTSLSTPGPSQK 4 NTRTIGGTAGSTAFKFTSLFTSGPTQK 5 STRVSGGGARPYHLGLHVPLYTWAISE

6 DTYVTGGAQGRTTRGFAGLFTSGPAQK 7 STRVTGEVQGHSLRGLTSFFASGPAQR 8 GTYTTGGAQGRATQGLASLFSRGSARK

Table 3 Amino acid sequence list of hepatitis C virus envelope protein HVR1 analog polypeptide

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.

SEQUENCE LISTING

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Claims (8)

1. a class hepatitis C virus envelope protein HVR1 mimic peptide is characterized in that this mimic peptide is arbitrary aminoacid sequence shown in the SEQ NO:2,4,6,8,10,12,14 or 16.

2. class hepatitis C virus envelope protein HVR1 simulation gene is characterized in that, the described mimic peptide of this simulation genes encoding claim 1.

3. simulation gene as claimed in claim 2 is characterized in that this simulation gene is an arbitrary nucleotide sequence shown in the SEQNO:1,3,5,7,9,11,13 or 15.

4. the application of mimic peptide as claimed in claim 1 is characterized in that the application of described mimic peptide in the medicine of preparation detection hepatitis C.

5. a carrier is characterized in that, it contains claim 2 or 3 described simulation genes.

6. a genetically engineered host cell is characterized in that, the host cell that it is transformed or transduce by the described carrier of claim 5.

7. preparation method who prepares hepatitis C virus envelope protein HVR1 mimic peptide is characterized in that this method comprises:

(a) be fit to express under the condition of hepatitis C virus envelope protein HVR1 mimic peptide the described host cell of cultivation claim 6:

(b) from culture, isolate hepatitis C virus envelope protein HVR1 mimic peptide.

8. a detection kit is characterized in that, it contains the described mimic peptide of claim 1.

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