CN118903475B - Application of recombinant protein rMmm604 in preventing infection of Mycoplasma mycoides subsp. - Google Patents
Application of recombinant protein rMmm604 in preventing infection of Mycoplasma mycoides subsp. Download PDFInfo
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Abstract
本发明公开了重组蛋白rMmm604在抗丝状支原体丝状亚种感染中的应用，本发明属于医药技术领域。本发明提出了一种新的抗丝状支原体丝状亚种感染的药物，该药物为重组蛋白rMmm604，研究显示，该重组蛋白rMmm604不会对EBL细胞产生细胞毒性，并且重组蛋白rMmm604孵育EBL细胞后，感染EBL的两种Mmm菌株（Ben1和Mu1）数量与对照组（标签蛋白rMBP孵育组）相比均明显减少。因此，本发明为牛传染性胸膜肺炎的治疗提供了新的技术手段。
The present invention discloses the application of recombinant protein rMmm604 in resisting infection of Mycoplasma mycoides subspecies, and the present invention belongs to the field of medical technology. The present invention proposes a new drug for resisting infection of Mycoplasma mycoides subspecies, which is recombinant protein rMmm604. Studies have shown that the recombinant protein rMmm604 does not produce cytotoxicity to EBL cells, and after incubating EBL cells with recombinant protein rMmm604, the number of two Mmm strains (Ben1 and Mu1) infected with EBL is significantly reduced compared with the control group (label protein rMBP incubation group). Therefore, the present invention provides a new technical means for the treatment of bovine contagious pleuropneumonia.
Description
Technical Field
The invention relates to a recombinant protein and application thereof in resisting filamentous mycoplasma subspecies infection. In particular to the application of recombinant protein rMmm to the resistance of mycoplasma filis subspecies infection. The invention belongs to the technical field of medicines.
Background
Infectious bovine pleuropneumonia (Contagious bovine pleuropneumonia, CBPP) is a severe respiratory disease caused by filamentous mycoplasma subspecies (Mycoplasma mycoides subsp. Mycoides, mmm) and susceptible to individual cattle. In the acute to subacute stage, the disease manifests as cellulosic bronchopneumonia and pleural effusion, and in chronic cases as lung abscess. CBPP is classified by the world animal health organization (World Organization for ANIMAL HEALTH, WOAH) as one of the six major diseases due to its high morbidity and mortality.
CBPP is an ancient disease, epidemic in central europe and northern europe since the lower half of the 16 th century. In the latter half of the 19 th century, CBPP began to spread worldwide with the increase in livestock commerce, with the first 20 th century being transferred into asia. To date, many countries eradicate CBPP by a post-kill or post-immunisation strategy, but remain popular in saharan africa. These areas are reported to have a total of up to 4480 kilo euros of direct and indirect economic losses due to CBPP annually, severely impacting local animal husbandry development. Some Asian countries lack effective monitoring data, and epidemic situation is unknown, but internal data show that the prevalence rate of the Bkenstein Ovap province reaches 8%. CBPP was introduced when the dairy cow was introduced in 1919, and thereafter, it was popular in our country for up to 70 years, and huge losses were caused to agriculture that was currently mainly relying on animal power to develop production activities. Since the 60 s of the 20 th century, china has been authenticated by WOAH to be CBPP-free country since no clinical cases were found after 1989 by high-density inoculation of an autonomously developed attenuated vaccine and combination of strict prevention and control measures. Because CBPP is still currently popular in the neighborhood of southeast asia and africa in our country, where the eastern, southern and western africa all experience large-scale bursts of CBPP. Under the new situation that trade is continuously aggravated at home and abroad nowadays, although CBPP is eradicated for years in China, the risk of re-entering and epidemic of the disease still exists. Notably, under the requirements of new national biosafety and animal welfare, the autonomous development of attenuated vaccines (rabbit/sheep hydrothorax) in China has been prohibited from production and use. Thus, there is a need for improvements and development of novel CBPP vaccines (e.g., subunit or nucleic acid vaccines, etc.), as well as for the development of safe and effective anti-infective agents.
Since 1956, the control and eradication of CBPP in domestic animals was successfully promoted by using an attenuated vaccine serial-passage cultured in allogeneic animals by Mmm virulent isolate Ben1, a Harbin veterinary institute of China academy of agricultural sciences. The laboratory was used as a national CBPP reference laboratory for whole genome sequencing of five representative strains (Ben 1, ben50, ben181, ben326 and Ben 468) passaged in the Ben series at a previous stage. Where Ben1 is a primary virulent strain, ben50 is a first generation strain that is virulent to rabbits, ben181 and Ben326 are vaccine strains, and Ben468 is a first generation strain lacking immune protection. The results of the comparison and sequencing revealed that the number of genes from Ben1 to Ben468,679 remained unchanged, constituting the core genes of Ben series, but 35 genes were lost in Ben 468. We speculate that the 35 gene expressed proteins may be associated with resistance to Mmm infection and immune protection.
Mmm604 is one of the 35 genes deleted, which expresses a putative protein of 154 amino acids in size, belonging to the ABC three-component system of proteins. Therefore, we tested its role in anti Mmm and put forward the present invention.
Disclosure of Invention
The invention aims to provide recombinant protein rMmm and application thereof in resisting mycoplasma filis subspecies infection.
In order to achieve the above purpose, the invention adopts the following technical means:
The invention firstly constructs Mmm prokaryotic expression plasmid and purifies recombinant protein rMmm with MBP label. EBL cells were incubated with 1. Mu.g/mL recombinant protein rMmm or tag protein rMBP for 2 hours, followed by the addition of 10 7 CCU/mL of Mmm two virulent strains (Ben 1 and Mu 1) per well for a further incubation of 12 h. After the incubation time was completed, the cell wells were washed 3 times with PBS and all EBL cells and Mmm adhered to their surfaces were collected. The relative level of change in the amount of Mmm adhering to EBL cells was analyzed and calculated using the present laboratory specific TaqMan probe. The results showed that after incubation of recombinant protein rMmm604,604 protein, the number of adhesion of the two Mmm strains (Ben 1 and Mu 1) to EBL was reduced compared to the control (tag protein rMBP incubation). The green fluorescence value (which represents Mmm signal) on the EBL cells treated as described above was examined by IFA, and the result showed that the number of EBL infected by both Mmm strains was reduced compared to the control group (tagged protein rMBP incubated group) after incubation of recombinant protein rMmm604. The above results demonstrate that recombinant protein rMmm604,604 is capable of inhibiting Mmm adhesion and infection to host EBL cells.
Based on the above research, the invention provides the application of recombinant protein rMmm to the resistance of mycoplasma filis subspecies infection.
Wherein, preferably, the nucleotide sequence of the recombinant protein rMmm is shown as SEQ ID NO.1 or SEQ ID NO. 2.
Preferably, the recombinant protein rMmm is obtained by cloning the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 into a prokaryotic expression plasmid, and then expressing and purifying by a prokaryotic expression system.
Preferably, the recombinant protein rMmm is provided with an MBP tag.
Wherein, preferably, the prokaryotic expression system is an escherichia coli expression system.
Wherein, preferably, the prokaryotic expression plasmid is pMAL-c5X.
Wherein, preferably, the mycoplasma filis subspecies comprise Ben1 and Mu1.
Compared with the prior art, the invention has the beneficial effects that:
The invention provides a novel medicament for resisting filamentous mycoplasma subspecies infection, which is recombinant protein rMmm604,604, and researches show that the recombinant protein rMmm604,604 does not generate cytotoxicity to EBL cells, and after the recombinant protein rMmm is used for incubating the EBL cells, the number of two Mmm strains (Ben 1 and Mu 1) infected with the EBL is obviously reduced compared with that of a control group (a tagged protein rMBP incubation group). Therefore, the invention provides a new technical means for treating the infectious bovine pleuropneumonia.
Drawings
FIG. 1 shows the PCR identification of recombinant plasmid pMAL-c5X-Mmm 604;
the left lane 1 is DNA MARKER, and the lanes 2-6 are the PCR amplification results of the recombinant plasmid pMAL-c 5X-Mmm;
FIG. 2 is a map of recombinant plasmid pMAL-c5X-Mmm604 after sequencing;
FIG. 3 shows the SDS-PAGE identification of prokaryotic expressed and purified recombinant protein rMmm;
FIG. 4 shows cytotoxicity assays after incubation of EBL cells with recombinant protein rMmm;
FIG. 5 is a graph showing the relative levels of change in the number of two virulent strains (Ben 1 and Mu 1) of Mmm attached to EBL cells after incubation of the host EBL cells with rMmm604 protein, as determined by the TAQMAN QPCR method;
Wherein A is a relative change level diagram of the number of Ben1, B is a relative change level diagram of the number of Mu 1;
FIG. 6 is a graph of green fluorescence results of IFA assay to determine Mmm infection with two virulent strains (Ben 1 and Mu 1) of recombinant protein rMmm after incubation of host EBL cells;
wherein A is a green fluorescence result graph of Ben1 infection, and B is a green fluorescence result graph of Mu1 infection.
Detailed Description
The invention is further described below in connection with specific examples which are given solely for illustration of the invention and are not intended to limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
EXAMPLE 1 prokaryotic expression and purification of recombinant protein rMmm604,604
1. Construction of recombinant plasmid pMAL-c5X-Mmm604,604
1.1 Obtaining Mmm nucleotide fragment:
Mmm604 (shown as SEQ ID NO. 1) and codon optimizing the sequence, wherein the optimized sequence is shown as SEQ ID NO.2, and Sac I and Not I enzyme cutting sites (the enzyme cutting site sequences are marked in an underlined form) are respectively added at two ends of the sequence shown as SEQ ID NO.2, and the sequence is shown as SEQ ID NO.3 by Beijing Hua big gene company.
1.2 Construction of pMAL-c5X-Mmm604 by T4 ligase method
The target sequence shown in SEQ ID No.3 was digested simultaneously with Sac I and Not I, and ligated overnight with the commercial prokaryotic plasmid pMAL-c5X (NEB, with MBP tag) via T4 ligase. The following day, the ligation products were transformed into DH5 alpha E.coli and plated for growth, and after single colonies were grown, single colonies were picked up, shaken and plasmids were extracted. The plasmid was then amplified by PCR to a fragment of the target size (474 bp) (FIG. 1), and the recombinant plasmid pMAL-c5X-Mmm was successfully obtained as determined by the Jilin Kumei company sequencing assay, and the map of the recombinant plasmid pMAL-c5X-Mmm is shown in FIG. 2.
2. Prokaryotic expression and purification of recombinant protein rMmm604,604
The recombinant plasmid pMAL-C5X-Mmm604 is transformed into competent cells of escherichia coli BL21 (DE 3), when the culture is carried out at 37 ℃ of 200 r/min until the OD600nm value reaches 0.4-0.6, IPTG with the final concentration of 0.5 mmol/L is added into bacterial liquid, and the induced expression of 10 h is continued at 37 ℃ of 200 r/min. The cells were collected by centrifugation and washed 3 times with PBS, the cells were resuspended by Column Buffer, and after sonication and centrifugation, the supernatant and pellet were collected, respectively, and the expression form of recombinant protein rMmm was analyzed by SDS-PAGE gel electrophoresis. The results indicated rMmm604,604 as a soluble expressed protein. The target protein was then purified by affinity chromatography using Amylose resin (Neb corporation), concentrated using a ultrafiltration tube, and analyzed by SDS-PAGE gel electrophoresis, which showed that the recombinant protein rMmm (FIG. 3) was obtained in very high purity. Finally, the BCA protein concentration determination kit is used for determining the concentration of the recombinant protein rMmm.
Example 2 cytotoxicity detection of recombinant protein rMmm604 against EBL cells example 2 cytotoxicity detection of recombinant protein rMmm604 against EBL cells
Fetal bovine lung epithelial cells (EBL) were counted and inoculated into 96-well cell culture plates (2000/well) and incubated with 1 μg of tag protein rMBP (laboratory save) and recombinant protein rMmm604 12h, respectively. According to the instructions of CCK-8 assay kit (Nanjinopran), 10. Mu.l of CCK-8 Solution was added to each well and incubation in incubator was continued for 1-4 h. And finally detecting the absorbance at 450 nm by using an enzyme-labeled instrument. CCK-8 assay showed that neither the tagged protein rMBP nor recombinant protein rMmm were cytotoxic to EBL cells when incubated (FIG. 4).
EXAMPLE 3 recombinant protein rMmm detection of Mmm adhesion/infection level after incubation of host EBL cells with recombinant protein rMmm
1. TAQMAN QPCR method for detecting Mmm adhesion level in EBL cells after incubation of recombinant protein rMmm604 (P4 laboratory procedure)
EBL cells were counted and plated into 12-well cell culture plates (3.2x10 5/well). The next day, cells in the cell wells grew almost full, 1 μg of tagged protein rMBP or recombinant protein rMmm, 604 2, h per well of cells, followed by the addition of 10 7 CCU/mL of Mmm two virulent strains (Ben 1 and Mu 1) per well for continued incubation 12 h. After the incubation time is over, the cell wells are washed 3 times with PBS, all cells are scraped off, and Mmm genomic total DNA is extracted by using OMEGA EZNA-cube bacterial DNA kit. The relative level of change in the amount of Mmm adhering to EBL cells was analyzed and calculated using the present laboratory specific TaqMan probe.
TAQMAN QPCR the results of the assay are shown in FIG. 5, which shows that after incubation of recombinant protein rMmm604, the number of adhesion of the two Mmm strains (Ben 1 and Mu 1) to EBL was reduced compared to the control (the tagged protein rMBP incubation).
2. Indirect Immunofluorescence (IFA) method for detecting Mmm infection level in EBL cells after incubation of recombinant protein rMmm (P4 laboratory procedure)
EBL cells were treated by the previous procedure, after the incubation time was over, the wells were washed 3 times with PBS, fixed with 4% paraformaldehyde 30min, blocked with PBS containing 5% fish gelatin 30min at room temperature, the monoclonal antibody 1D9 of mouse antibody Mmm (prepared and stored in laboratory) was diluted 1:200 in PBS, incubated overnight at 4 ℃ for the cells, washed 3 times with PBS the next day, 10min each time, followed by co-incubation of cells with goat anti-mouse IgG-Alexa Fluor 488 fluorescent secondary antibody (1:500; invitrogen), incubated 30min at room temperature in the absence of light, washed three times with PBS each time 5min, and stained nuclei with DAPI (4', 6-diamino-2-phenylindole) (1:1000; sigma, usa). The PBS was washed three times, and the fluorescence level was observed using a fluorescence microscope and photographed.
The results of IFA detection of green fluorescence values are shown in fig. 6, which shows that after incubation of recombinant protein rMmm, the number of two Mmm strains (Ben 1 and Mu 1) infected with EBL was reduced compared to the control group (tag protein rMBP incubation group).
Claims (6)
1. The application of the recombinant protein rMmm in preparing medicaments for resisting infection of filamentous mycoplasma subspecies (Mycoplasma mycoides subsp. Mycides, mmm) is disclosed, and the nucleotide sequence for encoding the recombinant protein rMmm604 is shown as SEQ ID NO.1 or SEQ ID NO. 2.
2. The use according to claim 1, wherein the recombinant protein rMmm is obtained by cloning the sequence shown in SEQ ID No.1 or SEQ ID No.2 into a prokaryotic expression plasmid, and then expressing and purifying by a prokaryotic expression system.
3. The use of claim 1, wherein the recombinant protein rMmm is MBP tagged.
4. The use according to claim 2, wherein the prokaryotic expression system is an e.
5. The use according to claim 2, wherein the prokaryotic expression plasmid is pMAL-c5X.
6. The use of claim 1, wherein said filamentous mycoplasma subspecies comprise Ben1 and Mu1.
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| Application Number | Priority Date | Filing Date | Title |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101217974A (en) * | 2005-07-07 | 2008-07-09 | 伯尔尼大学 | Mycoplasma subunit vaccine |
| CN118161631A (en) * | 2024-05-08 | 2024-06-11 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Application of Ifi47 overexpression plasmid in resisting mycoplasma filiform subspecies infection |
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| DE60025522T2 (en) * | 1999-11-29 | 2006-07-06 | Akzo Nobel N.V. | MYCOPLASMA MYCOIDES SUBSP. MYCOIDES SC ANTIGENIC LPPQ PROTEIN, ITS MANUFACTURE AND USE |
| CN1300318C (en) * | 2004-08-05 | 2007-02-14 | 中国农业科学院哈尔滨兽医研究所 | DNA which code Mycoplasma mycoides subspecies N terminal lipoprotein |
| WO2017011919A1 (en) * | 2015-07-22 | 2017-01-26 | University Of Saskatchewan | Mycoplasma vaccines and uses thereof |
| CN116773806B (en) * | 2023-04-14 | 2026-01-02 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Competitive ELISA Detection Kit for Bovine Infectious Pleuropneumonia and Its Application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101217974A (en) * | 2005-07-07 | 2008-07-09 | 伯尔尼大学 | Mycoplasma subunit vaccine |
| CN118161631A (en) * | 2024-05-08 | 2024-06-11 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Application of Ifi47 overexpression plasmid in resisting mycoplasma filiform subspecies infection |
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