Application of recombinant protein rMmm604 in preventing infection of Mycoplasma mycoides subsp.

Abstract

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

[0001] The present invention relates to a recombinant protein and its application in resisting infection of Mycoplasma mycoides subspecies, and particularly to the application of recombinant protein rMmm604 in resisting infection of Mycoplasma mycoides subspecies. The present invention belongs to the field of medical technology. Background Art

[0002] Contagious bovine pleuropneumonia (CBPP) is a bacterial infection caused by Mycoplasma mycoides subsp. Mycoplasma mycoides subsp. mycoides CBPP is a severe respiratory disease caused by bronchitis (Mmm) in cattle of all breeds. In the acute to subacute stages, the disease presents as fibrinous bronchopneumonia and pleural effusion, while in chronic cases it presents as lung abscesses. Due to its high morbidity and mortality, CBPP is listed as one of the six priority diseases by the World Organization for Animal Health (WOAH).

[0003] CBPP is an ancient disease that has been prevalent in Central and Northern Europe since the second half of the 16th century. In the second half of the 19th century, with the increase in livestock trade, CBPP began to spread globally and was introduced to Asia in the early 20th century. To date, many countries have eradicated CBPP through culling or immunization and then culling strategies, but it is still prevalent in sub-Saharan Africa. It is reported that the direct and indirect economic losses caused by CBPP in these regions total up to 44.8 million euros each year, which seriously affects the development of local animal husbandry. Due to the lack of effective monitoring data, the epidemic situation in some Asian countries is unknown, but according to internal data, the prevalence rate in Punjab Province, Pakistan reached 8%. CBPP was introduced to my country when dairy cows were introduced in 1919. Since then, it has been prevalent in my country for 70 years, causing huge losses to agriculture, which mainly relied on animal power for production activities at that time. Since the 1960s, my country has adopted high-density vaccination of independently developed attenuated vaccines combined with strict prevention and control measures. Since 1989, no clinical cases have been found. In 2011, it was certified as a CBPP-free country by WOAH. Since CBPP is still prevalent in Southeast Asia and Africa, which are adjacent to my country, East, South and West Africa have all experienced large-scale outbreaks of CBPP. Under the new situation of increasing domestic and foreign trade, although my country has eradicated CBPP for many years, it still faces the risk of re-introduction and prevalence of the disease. It is worth noting that under the new national biosafety law and animal welfare requirements, the attenuated vaccine (rabbit / lamb pleural effusion vaccine) independently developed by my country has been banned from production and use. Therefore, we urgently need to improve and develop new CBPP vaccines (such as subunit or nucleic acid vaccines, etc.), as well as develop safe and effective anti-infective drugs.

[0004] Since 1956, the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences has successfully promoted the control and eradication of CBPP in livestock in my country by using the attenuated vaccine produced by continuous passage of the virulent isolate Ben1 of Mmm in allogeneic animals. As a national CBPP reference laboratory, our laboratory has previously performed whole genome sequencing on five representative rabbit-passaged strains (Ben1, Ben50, Ben181, Ben326, and Ben468) of the existing Ben series. Among them, Ben1 is the primary virulent strain, Ben50 is the first generation strain that is toxic to rabbits for the first time, Ben181 and Ben326 are vaccine strains, and Ben468 is a first generation strain that lacks immune protection. Comparison of sequencing results shows that from Ben1 to Ben468, 679 genes remain unchanged, constituting the core genes of the Ben series, but 35 genes are lost in Ben468. We speculate that the proteins expressed by these 35 genes may be related to anti-Mmm infection and immune protection.

[0005] Mmm604 It is one of the 35 deleted genes, which expresses a hypothetical protein of 154 amino acids, belonging to the ABC three-component system protein. Therefore, we tested its role in anti-Mmm and proposed the present invention. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant protein rMmm604 and its application in resisting infection of Mycoplasma mycoides subspecies.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention first constructs Mmm60 4 prokaryotic expression plasmid and purified the recombinant protein rMmm604 with MBP tag. EBL cells were incubated with 1 μg / ml recombinant protein rMmm604 or tagged protein rMBP for 2 hours, and then 10 7CCU / mL of two strong strains of Mmm (Ben1 and Mu1) were incubated for 12 h. After the incubation time was over, the cell wells were washed 3 times with PBS, and all EBL cells and Mmm adhered to their surfaces were collected. The relative change level of the number of Mmm adhered to EBL cells was analyzed and calculated using the specific TaqMan probe of this laboratory. The results showed that after incubation with the recombinant protein rMmm604 protein, the number of two Mmm strains (Ben1 and Mu1) adhering to EBL was reduced compared with the control group (tag protein rMBP incubation group). The green fluorescence value (the green fluorescence represents the Mmm signal) on the EBL cells treated above was detected by IFA. The results showed that after incubation with the recombinant protein rMmm604, the number of EBL infected by the two Mmm strains was reduced compared with the control group (tag protein rMBP incubation group). The above results indicate that the recombinant protein rMmm604 can inhibit the adhesion and infection of Mmm to host EBL cells.

[0009] On the basis of the above research, the present invention proposes the use of recombinant protein rMmm604 in resisting infection of Mycoplasma mycoides subspecies.

[0010] Among them, preferably, the nucleotide sequence encoding the recombinant protein rMmm604 is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0011] Among them, preferably, the recombinant protein rMmm604 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 it through a prokaryotic expression system.

[0012] Among them, preferably, the recombinant protein rMmm604 carries an MBP tag.

[0013] Among them, preferably, the prokaryotic expression system is an Escherichia coli expression system.

[0014] Among them, preferably, the prokaryotic expression plasmid is pMAL-c5X.

[0015] Among them, preferably, the mycoplasma mycoides subspecies include Ben1 and Mu1.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention proposes a new drug for resisting infection of Mycoplasma mycoides subspecies, which is a recombinant protein rMmm604. Studies have shown that the recombinant protein rMmm604 does not produce cytotoxicity to EBL cells, and after incubating EBL cells with the 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The recombinant plasmid pMAL-c5X- Mmm604 PCR identification results;

[0019] From left: Lane 1 is DNA Marker; Lanes 2-6 are recombinant plasmids pMAL-c5X- Mmm604 PCR amplification results;

[0020] Figure 2 The recombinant plasmid pMAL-c5X- Mmm604 Post-sequencing profile;

[0021] Figure 3 The SDS-PAGE identification results of prokaryotic expression and purification of recombinant protein rMmm604;

[0022] Figure 4 Cytotoxicity test after incubation of EBL cells with recombinant protein rMmm604;

[0023] Figure 5 This is a graph showing the relative changes in the number of two strong strains of Mmm (Ben1 and Mu1) attached to EBL cells after incubation of host EBL cells with rMmm604 protein using TaqMan qPCR analysis;

[0024] Among them, A is the relative change level diagram of Ben1 quantity; B is the relative change level diagram of Mu1 quantity;

[0025] Figure 6 This is the green fluorescence result of the infection of two strong strains of Mmm (Ben1 and Mu1) after the host EBL cells were incubated with the recombinant protein rMmm604 using the IFA method;

[0026] Among them, A is the green fluorescence result of Ben1 infection; B is the green fluorescence result of Mu1 infection. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific embodiments, but the embodiments are only used to illustrate the present invention and do not constitute any limitation to the protection scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0028] Example 1 Prokaryotic expression and purification of recombinant protein rMmm604

[0029] 1. Construction of recombinant plasmid pMAL-c5X-Mmm604

[0030] 1.1 Obtaining the Mmm604 nucleotide fragment:

[0031] The original sequence of Mmm604 is shown in SEQ ID NO.1. The sequence was codon-optimized and the optimized sequence is shown in SEQ ID NO.2. Sac I and Not I restriction sites were added to both ends of the sequence shown in SEQ ID NO.2 (the restriction site sequences are marked with underscores). The sequence was obtained from Beijing BGI Genomics Co., Ltd. and the sequence is shown in SEQ ID NO.3.

[0032] 1.2 Construction of pMAL-c5X-Mmm604 using T4 ligase method

[0033] The target sequence shown in SEQ ID NO.3 was double-digested with Sac I and Not I and commercial prokaryotic plasmid pMAL-c5X (NEB, with MBP tag) and then ligated overnight with T4 ligase. The next day, the ligation product was transformed into DH5α E. coli and plated for growth. After a single colony was grown, it was picked, shaken and the plasmid was extracted. The plasmid was then amplified by PCR to obtain the target size fragment (474 ​​bp) ( Figure 1 ), and sent it to Jilin Kumei Company for sequencing and identification to confirm that the recombinant plasmid pMAL-c5X-Mmm604 was successfully obtained. The map of the recombinant plasmid pMAL-c5X-Mmm604 is as follows Figure 2 shown.

[0034] 2. Prokaryotic expression and purification of recombinant protein rMmm604

[0035] The recombinant plasmid pMAL-c5X-Mmm604 was transformed into competent E. coli BL21 (DE3) cells and cultured at 37°C 200 r / min until the OD600nm value reached 0.4-0.6. IPTG with a final concentration of 0.5 mmol / L was added to the bacterial solution, and the expression was induced at 37°C 200 r / min for 10 h. The bacteria were collected by centrifugation and washed 3 times with PBS. The bacteria were resuspended in Column Buffer. After ultrasonic crushing and centrifugation, the supernatant and precipitate were collected respectively, and the expression form of the recombinant protein rMmm604 was analyzed by SDS-PAGE gel electrophoresis. The results showed that rMmm604 was a soluble expression protein. Subsequently, the target protein was purified by affinity chromatography using Amylose resin (Neb Company), and the target protein was concentrated using an ultrafiltration tube. SDS-PAGE gel electrophoresis analysis showed that a highly pure recombinant protein rMmm604 ( Figure 3 ). Finally, the concentration of recombinant protein rMmm604 was determined using the BCA protein concentration assay kit.

[0036] Example 2 Detection of cytotoxicity of recombinant protein rMmm604 on EBL cells Example 2 Detection of cytotoxicity of recombinant protein rMmm604 on EBL cells

[0037] Fetal bovine lung epithelial cells (EBL) were counted and inoculated into 96-well cell culture plates (2000 cells / well), and incubated with 1 μg of tagged protein rMBP (preserved in the laboratory) and recombinant protein rMmm604 for 12 h. According to the instructions for use of the CCK-8 detection kit (Nanjing Novogene), 10 μl of CCK-8 Solution was added to each well and incubated in the incubator for 1-4 h. Finally, the absorbance at 450 nm was detected using an ELISA reader. The CCK-8 test results showed that incubation with tagged protein rMBP and recombinant protein rMmm604 did not cause cytotoxicity to EBL cells ( Figure 4 ).

[0038] Example 3 Detection of Mmm adhesion / infection level after incubation of host EBL cells with recombinant protein rMmm604

[0039] 1. TaqMan qPCR method to detect the Mmm adhesion level in EBL cells after incubation with recombinant protein rMmm604 (P4 laboratory operation)

[0040] After EBL cells were counted, they were inoculated into 12-well cell culture plates (3.2*10 5 The next day, when the cells in the wells were almost full, 1 μg of the tagged protein rMBP or recombinant protein rMmm604 was incubated in each well for 2 h, and then 10 7CCU / mL of two strong strains of Mmm (Ben1 and Mu1) were incubated for 12 h. After the incubation time was over, the cell wells were washed 3 times with PBS, all cells were scraped with a cell spatula, and the total genomic DNA of Mmm was extracted using the OMEGA EZNA® Bacterial DNA Kit. The relative change level of the number of Mmm attached to EBL cells was analyzed and calculated using the laboratory-specific TaqMan probe.

[0041] TaqMan qPCR test results are as follows Figure 5 As shown, the results showed that after incubation with the recombinant protein rMmm604, the number of two Mmm strains (Ben1 and Mu1) adhering to EBL was reduced compared with the control group (incubation group with the tag protein rMBP).

[0042] 2. Indirect immunofluorescence (IFA) method to detect the Mmm infection level in EBL cells after incubation with recombinant protein rMmm604 (P4 laboratory operation)

[0043] EBL cells were treated with the above steps. After the incubation time, the cell wells were washed with PBS three times, fixed with 4% paraformaldehyde for 30 min, blocked with PBS containing 5% fish gelatin for 30 min at room temperature, diluted with mouse anti-Mmm monoclonal antibody 1D9 (prepared and stored in the laboratory) at 1:200 in PBS, and incubated at 4°C overnight. The next day, the cells were washed with PBS three times, 10 min each time. The cells were then incubated with goat anti-mouse IgG-Alexa Fluor 488 fluorescent secondary antibody (1:500; Invitrogen) at room temperature in the dark for 30 min, washed three times with PBS for 5 min each time, and stained with DAPI (4',6-diamidino-2-phenylindole) (1:1000; Sigma, USA). The cells were washed with PBS three times, and the fluorescence level was observed using a fluorescence microscope and photographed.

[0044] The results of IFA green fluorescence detection are as follows Figure 6 As shown, the results showed that after incubation with the recombinant protein rMmm604, the number of two Mmm strains (Ben1 and Mu1) infected with EBL was reduced compared with the control group (incubation group with the tag protein rMBP).

Claims

1. Use of the recombinant protein rMmm604 in the preparation of a drug for resisting infection by Mycoplasma mycoides subsp. mycoides (Mmm), wherein the nucleotide sequence encoding the recombinant protein rMmm604 is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The recombinant protein rMmm604 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 it through a prokaryotic expression system.

3. The use according to claim 1, characterized in that The recombinant protein rMmm604 carries an MBP tag.

4. The use according to claim 2, characterized in that The prokaryotic expression system is an Escherichia coli expression system.

5. The use according to claim 2, characterized in that The prokaryotic expression plasmid is pMAL-c5X.

6. The use according to claim 1, characterized in that The mycoplasma mycoides subspecies include Ben1 and Mu1.

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