Mycoplasma hyopneumoniae recombinant antigen, expression vector, recombinant bacterium and application

Abstract

The invention belongs to the technical field of medical biology, and particularly discloses a mycoplasma hyopneumoniae recombinant antigen, an expression vector, a recombinant bacterium and application. The invention provides a construction method of a mycoplasma hyopneumoniae recombinant antigen, which is characterized in that a mycoplasma hyopneumoniae P97 protein R1 region is modified to construct the mycoplasma hyopneumoniae recombinant antigen as shown in SEQ ID No.1. The mycoplasma hyopneumoniae recombinant antigen is cloned into an expression vector, and a recombinant engineering bacterium is obtained by using a genetic transformation means. The recombinant engineering bacterium can stably express the recombinant antigen of the mycoplasma hyopneumoniae, can be prepared into an oral living bacterium preparation, has the characteristics of safety, effectiveness, rapidness and the like, and particularly can prevent infection of the mycoplasma hyopneumoniae to pigs without an immune response process.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a recombinant antigen, an expression vector and a recombinant bacterium of Mycoplasma hyopneumoniae and applications thereof. Background Art

[0002] Mycoplasma hyopneumoniae (Mhp) is a pathogen that causes Mycoplasma pneumonia (MPS) in pigs, which is a chronic respiratory disease. It is also called swine asthma and swine endemic pneumonia. Generally, pigs are susceptible to MPS and can be infected at all ages. It can cause coughing, asthma, anorexia and other symptoms in pigs. Clinically, it manifests as reduced daily weight gain and obvious lung lesions in piglets, which causes huge economic losses to the pig industry.

[0003] When pigs are naturally infected with Mycoplasma pneumoniae, they can also develop immunity. However, due to the poor immunogenicity of Mycoplasma, it takes a long time to obtain effective immune protection. The method of obtaining immunity through infection is less effective and cannot protect fattening pigs from MPS. Domestic and foreign studies have shown that it is very difficult to completely kill the pathogen after the occurrence of wheezing disease, especially for respiratory infections, because it is difficult for disinfectants to penetrate into the surface of the lung mucosa to work; at the same time, antibiotic treatment cannot repair the damage caused. If disinfection is stopped, the pathogen will escape again and the spread of the disease will start again, causing heavy losses to the farm. Summary of the invention

[0004] The purpose of the present invention is to provide a recombinant antigen, expression vector and recombinant bacteria of Mycoplasma hyopneumoniae and their applications, to construct a Lactococcus lactis expression system for expressing Mycoplasma hyopneumoniae P97 protein, and to prepare an oral live bacterial preparation for preventing Mycoplasma hyopneumoniae infection, which can prevent Mycoplasma hyopneumoniae infection without the need for an immune response process.

[0005] The present invention provides a method for constructing a recombinant antigen of Mycoplasma hyopneumoniae, comprising using the Mycoplasma hyopneumoniae P97 gene as a reference gene and modifying the C-terminus of the reference gene;

[0006] The modification includes connecting the R1 region containing 12 repeating motifs at the C-terminus of the reference gene in series three times with a flexible connecting peptide, and adding a His tag at the C-terminus;

[0007] The GenBank accession number of the Mycoplasma hyopneumoniae P97 gene is AY512904.1.

[0008] The present invention also provides a recombinant Mycoplasma hyopneumoniae antigen constructed by the above construction method.

[0009] Preferably, the nucleotide sequence of the recombinant Mycoplasma hyopneumoniae antigen is shown as SEQ ID No.1.

[0010] The present invention also provides a recombinant vector comprising the above-mentioned Mycoplasma hyopneumoniae recombinant antigen.

[0011] The present invention also provides a recombinant bacterium comprising the recombinant vector.

[0012] Preferably, the recombinant bacteria uses Lactococcus lactis as a basic vector.

[0013] The present invention also provides a fermentation liquid containing live bacteria obtained after fermentation of the recombinant bacteria.

[0014] The present invention also provides an oral live bacteria preparation for preventing Mycoplasma hyopneumoniae infection, wherein the oral live bacteria preparation uses the fermentation liquid as an effective ingredient.

[0015] Preferably, the number of live bacteria in the oral live bacteria preparation is (0.8-1.2)×10 8 cfu / mL.

[0016] Beneficial effects: The present invention provides a method for constructing a recombinant antigen of Mycoplasma hyopneumoniae, wherein the R1 region of the Mycoplasma hyopneumoniae P97 protein is modified to construct a recombinant antigen of Mycoplasma hyopneumoniae shown in SEQ ID No. 1. The present invention also clones the recombinant antigen of Mycoplasma hyopneumoniae into an expression vector, and obtains a recombinant engineered bacterium by genetic transformation.

[0017] The recombinant engineered bacteria of the present invention can stably express the recombinant antigen of Mycoplasma hyopneumoniae, which can be prepared into an oral live bacterial preparation. Compared with vaccines, the oral preparation created by the present invention is safer, more effective and faster. The safety of the oral preparation of the present invention is reflected in the small molecule functional protein of the effective ingredient, and the absence of pathogenic genes; the effectiveness is reflected in that the effective ingredient of the oral preparation only secretes protective antigens, acts on the mucosal part of the body surface, and seizes the receptors of the target cells of Mycoplasma hyopneumoniae. When mycoplasma invades, the site where it binds to the mucosal surface cells is blocked and closed, thereby blocking the mycoplasma infection path; the rapidity is reflected in that the secretory protein expressed by Lactococcus lactis directly seizes the receptors that bind to the pathogen and the target cell, thereby blocking the mycoplasma infection, and no immune response process is required.

[0018] In the embodiment of the present invention, after the pig takes the preparation, the antigen protein (P97 protein) secreted by Lactococcus lactis in the preparation adheres to the respiratory mucosal mycoplasma target cell receptor, blocks the mycoplasma receptor protein site on the mucosal surface, and plays a role in ecological occupation. When mycoplasma in the environment invades the body, the mycoplasma binding site on the target cell has been completely blocked by the antigen protein biofilm, and the mycoplasma cannot bind to the mycoplasma binding site on the target cell, thereby effectively blocking the mycoplasma from binding to the receptor on the cell surface, thereby preventing Mycoplasma hyopneumoniae infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The plasmid structure diagram of the recombinant vector pNZ8149-P97 of the present invention;

[0020] Figure 2 This is a Western Blot verification diagram for verifying the expression of P97 protein in the present invention; in the figure, 1: total protein of pNZ8149-P97 / NZ3900 strain; 2: precipitation of pNZ8149-P97 / NZ3900 strain; 3: total protein of pNZ8149 / NZ3900 strain; M: protein marker;

[0021] Figure 3 Western blot was used to verify the stability of P97 protein expression in the present invention. Blot verification diagram, in the diagram, M: protein marker; 1: total protein of pNZ8149 / NZ3900 strain; 2: 1st generation of pNZ8149-P97 / NZ3900 strain; 3: 2nd generation of pNZ8149-P97 / NZ3900 strain; 4: 3rd generation of pNZ8149-P97 / NZ3900 strain; 5: 4th generation of pNZ8149-P97 / NZ3900 strain; 6: 5th generation of pNZ8149-P97 / NZ3900 strain; 7: 6th generation of pNZ8149-P97 / NZ3900 strain; 8: 7th generation of pNZ8149-P97 / NZ3900 strain; 9: 8th generation of pNZ8149-P97 / NZ3900 strain. DETAILED DESCRIPTION

[0022] The present invention provides a method for constructing a recombinant antigen of Mycoplasma hyopneumoniae, comprising using the Mycoplasma hyopneumoniae P97 gene as a reference gene and modifying the C-terminus of the reference gene;

[0023] The modification includes connecting the R1 region containing 12 repeating motifs at the C-terminus of the reference gene in series three times with a flexible connecting peptide, and adding a His tag at the C-terminus;

[0024] The GenBank accession number of the Mycoplasma hyopneumoniae P97 gene is AY512904.1.

[0025] The P97 protein of the present invention is an adhesion factor of Mycoplasma hyopneumoniae, plays a key role in the pathogenic process, is located on the surface of the cell membrane, can specifically bind to the cilia of the pig respiratory mucosa, and cause the occurrence of asthma. The P97R1 region is the main antigenic determinant, which is directly involved in the region of Mhp adhesion to cells. The Mycoplasma hyopneumoniae P97 gene published in GenBank (AY512904.1) is used as a reference, the R1 region (AAKPE / AAKPV) containing 12 repeating motifs at the C-terminus of the gene sequence is connected three times in series with a flexible connecting peptide (GGGGS), and a His tag is added at the C-terminus, and the sequence is sent to Nanjing GenScript Biotech Co., Ltd. for optimization and synthesis.

[0026] The present invention also provides a recombinant Mycoplasma hyopneumoniae antigen constructed by the above construction method.

[0027] The nucleotide sequence of the recombinant Mycoplasma hyopneumoniae antigen of the present invention is modified and synthesized as described above, and its sequence is shown in SEQ ID No. 1:

[0028] .

[0029] The present invention also provides a recombinant vector comprising the above-mentioned Mycoplasma hyopneumoniae recombinant antigen.

[0030] The recombinant vector of the present invention is preferably based on the Lactococcus lactis expression vector. In the embodiment, pNZ8149 is used as the base vector for illustration, but it cannot be stabilized as the entire protection scope of the present invention. The present invention preferably clones the sequence described in SEQ ID No.1 into the Sph I and Xbal I restriction sites of pNZ8149 by double restriction digestion to form a recombinant vector pNZ8149-P97. The recombinant plasmid structure is as follows: Figure 1 shown.

[0031] The present invention also provides a recombinant bacterium comprising the recombinant vector.

[0032] The recombinant bacteria of the present invention preferably use Lactococcus lactis as a basic vector. The present invention also provides a method for constructing the recombinant bacteria, preferably comprising the following steps: (1) cloning the sequence shown in SEQ ID No.1 into the Sph I and Xbal I restriction sites of pNZ8149 to form a recombinant vector pNZ8149-P97;

[0033] (2) The pNZ8149-P97 recombinant plasmid is transformed into competent Lactococcus lactis to obtain recombinant Lactococcus lactis expressing Mycoplasma hyopneumoniae P97 protein. The present invention does not specifically limit the method of the transformation, and genetic transformation can be performed using conventional methods in the art. For example, in the embodiment, the recombinant plasmid is transformed into NZ3900 Lactobacillus casei competent cells by electroporation to obtain the recombinant Lactococcus lactis. The present invention utilizes the construction method to construct a recombinant Lactococcus lactis that secretes the recombinant Mycoplasma hyopneumoniae immunogenic protein P97.

[0034] The present invention also provides a fermentation liquid containing live bacteria obtained after fermentation of the recombinant bacteria.

[0035] The fermentation medium of the present invention preferably includes Lactococcus lactis GM17 liquid culture medium, the fermentation temperature is preferably 30° C., and the fermentation time is preferably 12 hours.

[0036] The present invention also provides an oral live bacteria preparation for preventing Mycoplasma hyopneumoniae infection, wherein the oral live bacteria preparation uses the fermentation liquid as an effective ingredient.

[0037] The number of live bacteria in the oral live bacteria preparation of the present invention is preferably (0.8-1.2)×10 8 cfu / mL, more preferably 1×10 8 cfu / mL.

[0038] In order to further illustrate the present invention, a recombinant Mycoplasma hyopneumoniae antigen, expression vector, recombinant bacteria and applications provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the test materials used in the examples of the present invention are all conventional commercial products in the art, such as restriction endonucleases Sph I and Xbal I, which were purchased from NEB, Taq enzyme, dNTP, DNA Marker DL15000, AgaroseGel DNA Purification Kit, and Mini BEST Plasmid Purification Kit, which were purchased from Dalian Bao Biotechnology Co., Ltd. The cloning vector pNZ8149 was provided by Beijing Baosai Biotechnology Co., Ltd. Nisin was purchased from Coollab Biotechnology Co., Ltd.

[0040] Example 1

[0041] 1. Entrust Nanjing GenScript Biotech Co., Ltd. to synthesize the sequence shown in SEQ ID No.1;

[0042] 2. Obtaining the recombinant expression vector pNZ8149-P97

[0043] The cloning vector pNZ8149 was ligated with the small fragment after double digestion with Sph I / Xbal I and the P97 gene fragment shown in SEQ ID No. 1 after the same double digestion, and the plasmid was extracted after electroporation and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing verification.

[0044] The recombinant plasmid was sequenced and compared with the inserted P97 gene fragment. The sequencing results were consistent with expectations, indicating that the synthesized P97 gene fragment was successfully inserted into the Lactobacillus vector pNZ8149, and the recombinant plasmid was successfully constructed. The positive plasmid was named pNZ8149-P97. Figure 1 shown.

[0045] 3. Preparation and detection of expression of Mycoplasma hyopneumoniae P97 gene

[0046] Electrotransformation of target gene in Lactobacillus NZ3900: The electrotransformed Lactococcus lactis NZ3900 was spread on an Eliker solid culture plate and cultured in a 30°C incubator for 48 hours. The colonies on the plate were picked for PCR verification, and the positive colonies were inoculated into a GM17 liquid culture medium and cultured at 30°C for 48 hours.

[0047] Primers used for PCR verification:

[0048] F(SEQ ID No.2):catgggtactgcaggcatgcACATGGCAGCAAAACCCGA

[0049] R(SEQ ID No.3):gaaagcttgagctctctagaTTAATGATGATGGTGGTGATGAACT

[0050] The recombinant strain was then verified by Western Blot for P97 protein. First, the bacterial liquid was collected and centrifuged at ultra-low temperature 8000r / min for 10 minutes to obtain the bacterial sludge. The supernatant was collected by ultrasonic disruption using an ultrasonic disruptor at 30% power, on for 6s, off for 5s, and 10min. An appropriate amount of loading buffer was added and SDS-PAGE electrophoresis was performed. After the electrophoresis, the membrane was transferred to a PVDF membrane at 80V for 80min. After transfer, the membrane was blocked with 0.2% gelatin for 2h, washed 3 times, and incubated overnight at 4°C with a specific polyclonal rabbit anti-P97 antibody (1:1000 dilution). The next day, the membrane was washed 3 times and incubated with HRP-labeled goat anti-rabbit IgG (1:5000 dilution) for 1h. After washing 3 times, the membrane was developed in a chemiluminescence imaging system.

[0051] Verification results are as follows Figure 2 As shown, the lactic acid bacteria recombinant strain has a single band at the predicted position, which is about 45 kDa, while the NZ3900 competent cell has no band, indicating that the recombinant strain pNZ8149-P97 has been transformed into the NZ3900 competent cell and expressed successfully.

[0052] Example 2

[0053] 1. Cultivation method of recombinant Lactococcus lactis expression system

[0054] The recombinant Lactococcus lactis expression system was inoculated into the Lactococcus lactis GM17 liquid culture medium at a 1% inoculation rate, and the fermentation liquid was harvested after 12 hours at 30°C.

[0055] 2. Live count of recombinant lactobacilli

[0056] Using the plate surface spreading method:

[0057] l. Take 9 sets of sterile plates and mark them with a marker pen. -4 , 10 -5 , 10 -6 (dilution) 3 sets each. Take another 6 test tubes containing 4.5mL of sterile water and label them 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 .

[0058] 2. Dilution Use a 1 mL sterile pipette to draw 1 mL of the thoroughly mixed Lactococcus lactis solution (test sample) and accurately place 0.5 mL to 10 -1 This is a 10-fold dilution. Add the excess bacterial solution back to the original bacterial solution. -1 Place the test tube on a test tube shaker to oscillate the bacterial solution thoroughly. Take another 1 mL pipette and insert it into the 10 -1 Pipette the bacterial suspension back and forth three times in the test tube to further disperse and mix the bacteria. -1 For 1 mL of bacterial solution, accurately place 0.5 mL to 10 -2 In the test tube, this is a 100-fold dilution, and the rest are similar.

[0059] 3. Take samples with three 1 mL sterile pipettes and take 10 -4 , 10 -5 and 10 -6 Place 1 mL of the diluted bacterial suspension into numbered sterile plates, with 0.2 mL in each plate.

[0060] 4. Prepare GM17 solid culture medium on a plate, and after autoclaving, pour it into a culture dish as soon as possible. After the culture medium solidifies, add 100 μL of the above-mentioned bacterial solution of different dilutions, spread it evenly with a sterile coating vessel, and then invert the plate in a 30°C constant temperature incubator for culture.

[0061] 5. Counting After 48 hours of culture, take out the culture plates, calculate the average number of colonies on three plates of the same dilution, and calculate according to the following formula: colony forming unit (cfu) per milliliter = average number of colonies in three replicates of the same dilution × dilution factor × 5.

[0062] Example 3

[0063] Stable Expression of Mycoplasma hyopneumoniae P97 Protein

[0064] 1. Test methods

[0065] The recombinant strain constructed in Example 1 was subjected to Western Blot verification for the stable expression of P97 protein. The pNZ8149-P97 recombinant strain was subcultured to the 8th generation, and the bacterial solution was collected and centrifuged at ultra-low temperature 8000r / min for 10min to obtain bacterial mud. The supernatant was collected by ultrasonic disruption with an ultrasonic disruptor at 30% power, on for 6s, off for 5s, and 10min. An appropriate amount of loading buffer was added and SDS-PAGE electrophoresis was performed. After the electrophoresis, the membrane was transferred to a PVDF membrane at 80V for 80min. After the membrane was transferred, it was blocked with 0.2% gelatin for 2h, washed 3 times, and incubated overnight at 4°C with a specific polyclonal rabbit anti-P97 antibody (1:1000 dilution). The next day, it was washed 3 times, and incubated for 1h with HRP-labeled goat anti-rabbit IgG (1:5000 dilution). After washing 3 times, it was placed in a chemiluminescence imaging system for development.

[0066] Western Blot was used to verify the stable expression of P97 protein in the recombinant strain. The verification results are as follows: Figure 3 As shown, the recombinant lactic acid bacteria strain has been stably expressed during the passage process, and the target band can be detected in the 8th generation, while the NZ3900 competent cell has no band. This shows that the recombinant strain pNZ8149-P97 can be stably expressed.

[0067] Example 4

[0068] The lactococci capable of secreting recombinant P97 protein prepared in Example 1 were diluted to 0.8-1.2×10 8 cfu / ml of bacterial liquid. The bacterial liquid was administered to SPF New Zealand rabbits and SD rats in the form of oral liquid, and a negative control group A was set up to orally take the same volume of normal saline, and a control group B was set up to orally take the same volume of GM17 culture liquid. Each group of New Zealand rabbits and SD rats had 4 animals each. After 2 weeks of observation, none of the 24 animals showed abnormal body temperature, allergies and other abnormal adverse phenomena. This shows that the lactococcus lactis that can secrete recombinant P97 protein prepared by the present invention is safe and has no side effects.

[0069] Example 5

[0070] There are about 300 pigs in a farm in Hengyang County, Hunan Province, with a body weight of about 20kg, divided into 2 groups. Before the experiment began, some pigs had already fallen ill, with symptoms such as panting and coughing. The farm conducted a recombinant lactic acid bacteria preparation test with a test period of 5 months. The experimental group was fed with a dose of 2.5mL / head once every 3 days, and lactic acid bacteria were added to the drinking water for oral administration. The pigs in the control group drank water normally without adding lactic acid bacteria preparations. The experimental and control groups were fed according to the various growth cycles. After the experiment, the pigs were marketed and the feed-to-meat ratio was calculated.

[0071] The test results are shown in Table 1. The number of pigs in the control group decreased from 150 to 95, 55 of them died, and the mortality rate reached 36.66%; the experimental group was already in the disease period before the test. After use, the number decreased from 150 to 122, 28 of them died, and the mortality rate was 18.66%; the test results show that the recombinant lactic acid bacteria preparation has a good effect.

[0072] Table 1 Comparison of the experimental effects of recombinant lactic acid bacteria

[0073]

[0074] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a recombinant antigen of Mycoplasma hyopneumoniae, characterized in that: The method comprises using the Mycoplasma hyopneumoniae P97 gene as a reference gene and modifying the C-terminus of the reference gene; The modification includes connecting the R1 region containing 12 repeating motifs at the C-terminus of the reference gene in series three times with a flexible connecting peptide, and adding a His tag at the C-terminus; The GenBank accession number of the Mycoplasma hyopneumoniae P97 gene is AY512904.

1.

2. The recombinant Mycoplasma hyopneumoniae antigen constructed by the construction method according to claim 1.

3. The recombinant Mycoplasma hyopneumoniae antigen according to claim 2, characterized in that: The nucleotide sequence of the recombinant Mycoplasma hyopneumoniae antigen is shown in SEQ ID No.

1.

4. A recombinant vector comprising the recombinant Mycoplasma hyopneumoniae antigen according to claim 2 or 3.

5. A recombinant bacterium comprising the recombinant vector according to claim 4.

6. The fermentation broth containing live bacteria obtained after fermentation of the recombinant bacteria according to claim 5.

7. An oral live bacterial preparation for preventing Mycoplasma hyopneumoniae infection, characterized in that: The oral live bacteria preparation uses the fermentation broth according to claim 6 as an active ingredient.

8. The oral live bacteria preparation according to claim 7, characterized in that: The number of live bacteria in the oral live bacteria preparation is (0.8-1.2)×10 8 cfu / mL.

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