CN111533791B

CN111533791B - Preparation method and application of leptospira adhesion protein rvWFA3-2

Info

Publication number: CN111533791B
Application number: CN202010333395.2A
Authority: CN
Inventors: 孙爱华; 严杰
Original assignee: Hangzhou Medical College
Current assignee: Hangzhou Medical College
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2022-02-15
Anticipated expiration: 2040-04-24
Also published as: CN111533791A

Abstract

A preparation method and application of leptospira adhesion protein rvWFA3-2, belonging to the technical field of molecular biology and immunology. A prokaryotic expression system of vWFA3-2 protein coding genes is constructed by a genetic engineering means, and recombinant protein rvWFA3-2 is obtained by induction and purification, and can be used for research and development of vaccines and research and development of leptospira infection antibody detection kits. According to the invention, bioinformatics analysis is adopted to confirm that a signal peptide secretory protein is arranged at the N terminal of the LA _4207 gene product, and the LA _4207 gene product contains a superfamily structural domain (34-200 aa). A novel method is provided for the in vitro preparation of the recombinant protein rvWFA 3-2. And proves that the prepared adhesion protein rvWFA3-2 plays a role in protecting the attack of pathogenic leptospira interrogans strains, generates high-titer protective antibodies and has a cross protection effect.

Description

Preparation method and application of leptospira adhesion protein rvWFA3-2

Technical Field

The invention belongs to the technical field of molecular biology and immunology, and particularly relates to a preparation method and application of leptospira adhesion protein rvWFA 3-2.

Background

The invasiveness determines whether the microorganisms can infect the host, and the infection is a prerequisite for pathogenesis, so the invasiveness becomes the key point for researching the pathogenic mechanism of pathogenic microorganisms in recent years. As is well known, the main material basis of invasiveness is invasive enzymes, adhesion is the first step of pathogenic microorganisms, and the essence of the adhesion is the process of binding pathogenic microorganism adhesion factor ligands with host cell receptors, so that pathogenic microorganisms can colonize in a host and then grow and reproduce to cause diseases.

Vaccination is the most effective and economical means for preventing and controlling infectious diseases. The genetic engineering vaccine is an important direction for vaccine research due to low production cost and good effect. The leptospira outer membrane protein is proved to have a genus-specific antigen, and the outer membrane proteins LipL21, LipL32, LipL41 and the like and epitope antigens thereof are also proved to have certain immunoprotection in previous researches, can be used as vaccine candidate antigens, and still cannot obtain satisfactory immune effects. The adhesion factor located in the outer membrane is closely related to the invasion of bacteria and can induce the organism to generate serum IgG antibody with immune protection function.

Hemophilia factor (von Willebrand factor, vWF) is a glycoprotein synthesized and secreted by vascular endothelial cells and megakaryocytes of humans and mammals, and can bind to platelet membrane glycoproteins GpIb and GpIIb-IIIa to induce platelet adhesion and aggregation. vWF is an important coagulation factor and has 12 functional domains, of which the a3 domain binds to basement membrane Collagen (COL) exposed after vascular injury to locally aggregate platelets, presumably related to adhesion function.

Disclosure of Invention

In view of the problems in the prior art, the invention discovers that the LA _4207 gene encoding product has vWFA3 region superfamily structural domain vWFA3-2 but no platelet GP-Ib receptor binding site through bioinformatics analysis, and the vWFA3-2 is presumed to play the role of an adhesion factor in the infection process of hook of question marks. A prokaryotic expression system of vWFA3-2 protein coding genes is constructed by adopting a genetic engineering means, recombinant protein rvWFA3-2 is obtained by induction and purification, and the function and application of the recombinant protein rWFA 3-2 are proved.

A preparation method of leptospira adhesion protein rvWFA3-2 is characterized by comprising the following steps:

(1) designing a PCR primer containing an endonuclease Nde I or Xho I enzyme cutting site for later use according to restriction endonuclease map analysis and signal peptide prediction results of a T-A cloning vector pET42a and multiple cloning sites of a T-A cloning vector and a subcloning vector LA _4207 gene of Leptospira interrogans;

(2) extracting the DNA of Leptospira interrogans lysine strain;

(3) obtained by the step (1)Amplifying a superfamily structural domain fragment in a coagulation factor A3 area in the LA _4207 gene by the PCR primer and the Leptospira interrogans lysine strain DNA obtained in the step (2) to obtain a target gene amplification fragment, detecting the amplification fragment by ethidium bromide pre-stained 1.5% agarose gel electrophoresis, connecting the target gene amplification fragment with pMD19-T by using a T-A cloning kit, and transforming the plasmid into competent E.coli DH5 alpha by using a calcium chloride method after overnight connection to form E.coli DH5 alpha^pMD19-T4207Extracting recombinant plasmid pMD19-T after screening blue and white⁴²⁰⁷Sequencing;

(4) the recombinant plasmid pMD19-T with correct target gene sequence of the sequencing result obtained in the step (3)⁴²⁰⁷The recombinant plasmid pET42a is obtained by digesting the expression vector pET42a with restriction endonucleases Nde I and Xho I, recovering the restriction endonucleases and connecting the restriction endonucleases under the action of T4 DNA ligase⁴²⁰⁷；

(5) pET42a is prepared by calcium chloride method⁴²⁰⁷Transformation into competent E.coli BL21DE3 to form E.coli BL21DE3^pET42a-4207The engineering strain is inoculated on a 50 mu g/mL kanamycin LB agar plate for isolated culture, 0.5mmol/L IPTG is used for induction to obtain the target recombinant protein rvWFA3-2, and the rvWFA3-2 is purified by adopting a Ni-NTA affinity chromatography column.

The preparation method of the leptospira adhesion protein rvWFA3-2 is characterized in that the nucleotide sequence of the upstream primer in the PCR primer in the step (1) is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.

The preparation method of the leptospira adhesion protein rvWFA3-2 is characterized in that the step (2) comprises the following specific steps: and centrifuging the Leptospira interrogans lysine strain culture at 12000r/min at 4 ℃ for 15min to obtain a precipitate, washing with sterilized PBS (0.01 mol/L) and pH7.2, centrifuging according to the method, repeating for 2 times to obtain a Leptospira interrogans precipitate, and extracting DNA by using a bacterial genome DNA preparation kit.

The preparation method of the leptospira adhesion protein rvWFA3-2 is characterized in that the amplification method in the step (3) is as follows: an EX-Taq high-fidelity PCR kit is adopted for carrying out amplification reaction, and PCR parameters are 94 ℃ for 5min, 94 ℃ for 30s, 50 ℃ for 30s, 72 ℃ for 90s, 30 cycles and 72 ℃ for 7 min.

The preparation method of the leptospira adhesion protein rvWFA3-2 is characterized in that the separation culture and IPTG induction method in the step (5) is as follows: shake culturing at 37 deg.C at 220r/min to A₆₀₀When the value is 0.6-0.8, 0.5mmol/L IPTG is added, and the culture is continued for 6h according to the method, so as to induce the expression of the target recombinant protein rvWFA 3-2.

The application of Leptospira adhesion protein rvWFA3-2 in preparing vaccine for treating Leptospira interrogans infection.

Application of Leptospira adhesion protein rvWFA3-2 in preparing a detection kit for Leptospira infection antibody.

The invention has the following beneficial effects: according to the invention, bioinformatics analysis is adopted to confirm that the N end of the LA _4207 gene product contains a signal peptide (1-20aa) secretory protein, and the LA _4207 gene product contains a superfamily structural domain (34-200aa) but does not contain a platelet GP-Ib receptor binding site. The invention discloses a new method for preparing recombinant protein rvWFA3-2 in vitro, and proves the adhesion factor characteristic of preparing recombinant protein rvWFA3-2, and rvWFA3-2 has protective effect on the attack of pathogenic leptospira interrogans strains, generates high-titer protective antibodies, and the generated antibodies have cross protection effect. The recombinant protein rvWFA3-2 prepared by the invention can be used for vaccine development and development of a leptospira infection antibody detection kit.

Drawings

FIG. 1 is a chart showing the result of bioinformatics analysis of LA _4207 gene product;

FIG. 2 is a graph showing the results of ELISA detection of rvWFA3-2 binding to hCO OL 1/3/4/6;

FIG. 3 is a graph showing SPR detection results of binding of rvWFA3-2 and hCOL 1/4;

FIG. 4 is a graph showing the increase in Lep4207-mRNA levels upon infection of cells with Leptospira interrogans lysine strain;

FIG. 5 is a graph showing enhancement of expression of Lep4207 upon infection of cells with Leptospira interrogans lysine strain.

Detailed Description

The invention will be further illustrated by the following specific examples and the accompanying drawings. In the embodiment, a prokaryotic expression system of vWFA3-2 protein coding genes is constructed by adopting a genetic engineering means, recombinant protein rvWFA3-2 is obtained by induction and purification, and the function and the effect of the recombinant protein rWFA 3-2 are proved by experiments.

Example (b):

materials (I) and (II)

1. Bacterial strain source and culture

The Leizhou icterohepatitis lysine strain, influenza typhoid influenza cold type 6 strain, autumn group autumn type 4 strain, Bomana Borna type Rous strain, Australia type 65-9 strain, seven day fever type 56069 strain, Canine type forest strain, Seluo type Wulff type L183 strain and Java type M10 strain are preserved by Zhejiang university pathogenic organism system, and the strains are recovered and subjected to subculture at 28 ℃ in EMJH culture medium. Coli BL21DE3 and e.coli DH5 α (Novagen) were subcultured in LB medium at 37 ℃.

2. Cell line origin and culture

Human umbilical vein endothelial cell line (HUVEC) and mouse vascular endothelial tumor cell line (EOMA) were purchased from cell banks of Shanghai cell biology institute of Chinese academy of sciences, and cultured in RPMI-1640 medium containing 10% Fetal Calf Serum (FCS) (GiBco), 100U/mL penicillin and 100. mu.g/mL streptomycin (Sigma) and DMEM high-sugar medium (GiBco) and 5% CO, respectively₂Subculturing at 37 ℃.

3. rLep-MauG, serum specimen and enzyme labeled antibody source

Leptospira interrogans recombinant cytochrome C peroxidase (rLep-MauG) was prepared from the university of Zhejiang pathogen lines. The rabbit anti-leptospira whole-bacterium serum is purchased from China pharmaceutical biologicals institute. HRP-labeled goat anti-human IgG and HRP-labeled goat anti-human IgM were purchased from Jackson ImmunoResearch, inc.

Second, method and results

1. Bioinformatics analysis

The product transmembrane region, signal peptide and functional domain of Leptospira interrogans lysine strain LA _4207 gene (GenBank access No.: NC-004342.2) were analyzed using TMHMM-2.0, SignalP-4.1 and NCBI-Batch CD-Search NCBI software.

Bioinformatics analysis results: the LA-4207 gene product has a signal peptide (1-20aa) secreted protein at the N-terminus, which contains a superfamily domain and a metal-ion-dependent adhesion site (MIDAS), as shown in FIG. 1.

2. PCR and sequencing of its products

According to restriction endonuclease map analysis and signal peptide prediction results of a Leptospira interrogans LA _4207 gene in GenBank, and T-A cloning and subcloning vector pET42a (Novagen) multiple cloning sites, PCR primers containing endonuclease Nde I or Xho I cleavage sites are designed and used for amplifying an mutextracellular superfamily structural region fragment of 576bp LA _4207 (52-627) gene without a signal peptide or a transmembrane region. Primers were synthesized by Shanghai Invitrogen corporation. LA _4207 gene upstream primer: 5 '-CGC CAT ATG (Nde I) GTA ACT GGA ACA AAC GTA TCT TCA-3', downstream primer: 5 '-CGC CTC GAG (Xho I) CCT AAG CGA ATC GAG AGC GGT ATA-3'. The nucleotide sequence of the lysine strain LA _4207 (52-627) of the icterohaemorrhagic group is shown in SEQ ID NO. 3.

Leptospira interrogans lysine strain culture was centrifuged at 12000r/min at 4 ℃ for 15min, and Leptospira interrogans pellet was washed with 0.01mol/L sterile PBS (pH7.2), centrifuged as above, and repeated 2 times, and the Leptospira interrogans pellet was DNA extracted using bacterial genomic DNA preparation kit (Axygen). The von Willebrand coagulation factor A3 region superfamily domain fragment in the LA _4207 gene was amplified using EX-Taq high fidelity PCR kit (TaKaRa). PCR parameters: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 50 ℃ for 30s, 72 ℃ for 90 s; 7min at 72 ℃. Detecting the amplified product by ethidium bromide pre-stained 1.5% agarose gel electrophoresis, respectively connecting the target gene amplified fragment with pMD19-T by using a T-A cloning kit (TaKaRa), connecting overnight, and transforming the target gene amplified fragment into E.coli DH5 alpha by using a calcium chloride method to form E.coli DH5 alpha^pMD19-T4207After screening blue and white, mutextracting T-A cloning plasmid pMD19-T by using bacterial plasmid preparation kit (Axygen)⁴²⁰⁷Sequencing was entrusted to Shanghai Invitrogen corporation.

3. Prokaryotic expression system construction and identification

Correctly sequenced pMD19-T⁴²⁰⁷And prokaryotic expression vector pET42a were digested with restriction endonucleases Nde I and Xho I (TaKaRa), recovered, ligated by T4 DNA ligase (TaKaRa), and the ligation product was transformed into a sense vectorFormation of e.coli BL21DE3 in e.coli BL21DE3 in state^pET42a-4207The engineered strain was then plated on 50. mu.g/mL kanamycin (Sigma) LB agar plates for isolation. Selecting single colony, adding into 50 μ g/mL kanamycin LB culture solution, extracting pET42a according to the above method⁴²⁰⁷And (6) sequencing again.

T-A cloning and subcloning results: the sequencing results of the PCR amplification product T-A clone and the subclone engineering bacteria mutextracted plasmid of the LA _4207 gene fragment are completely consistent with the mutexpectation.

4. Expression and purification of target recombinant protein

E.coli BL21DE3^pET42a-4207Inoculating to 50 mu g/mL kanamycin LB culture solution, shaking culturing at 37 ℃ at 220r/min until A₆₀₀When the value is 0.6-0.8, 0.5mmol/L IPTG (Sigma) is added, and the culture is continued for 6h according to the method, so as to induce the expression of the target recombinant protein rvWFA 3-2. rvWFA3-2 was purified using a Ni-NTA affinity chromatography column (BioColor), checked for purity by 15% gel SDS-PAGE, and protein concentration was determined using BCA protein quantitation kit (Thermo Scientific).

Expression and purification results of the recombinant synthetic protein: coli BL21DE3 was induced with IPTG at a final concentration of 0.5mmoL/L^pET42a-4207Then rvWFA3-2 can be effectively expressed. SDS-PAGE of the supernatant and the precipitate after the ultrasonic disruption of the thalli and the ultrasonically disrupted thalli purified Ni-NTA shows that a specific band with the size of about 22.1kDa exists in the supernatant, is soluble protein and is consistent with the expected molecular mass of the recombinant protein. The vWFA3-2 protein amino acid sequence is shown in SEQ ID NO. 4.

5. rvWFA-IgG preparation and potency assay

1mg of rvWFA3-2 and Freund's adjuvant are mixed well and injected into the back of rabbit at multiple points and in the same skin for 4 times, each time is separated by one week. Serum was isolated from heart blood at 15d after the last immunization. Serum IgG was extracted by saturated ammonium sulfate precipitation and DEAE-52 (Sigma) ion exchange chromatography, and the titer of rvWFA-2-IgG was determined by immunodouble diffusion.

results for rvWFA-IgG titers: the rvWFA-IgG titer was 1: 8. Indicating that the rvWFA3-2 has good antigenicity.

6. ELISA detection

Human type I, III, IV, VI collagen (hCOL1/3/4/6, Sigma) was dissolved in 0.5mol/L acetic acid-physiological saline and coated onto polystyrene 96-well plates (Costar) overnight at 4 ℃ with 2. mu.g of protein per well. The plate was washed 5 times with 0.05% Tween20-PBS, and then blocked with 3% FCS-PBS overnight at 4 ℃. After washing the plate as above, 100. mu.L of rvWFA3-2 was added, and the plate was washed after incubation at 37 ℃ for 1 h. The method comprises the steps of taking 1:200 diluted rvWFA3-2-IgG as a primary antibody, taking 1:5000 diluted horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (Abcam) as a secondary antibody, incubating at 37 ℃ for 2h, washing the plate, adding 100 mu L (Abcam) of TMB color development liquid into each hole, developing at room temperature in a dark place for 1h, adding 100 mu L of stop solution into each hole, and detecting the light absorption value (OD450) at 450nm by using an M5 type multifunctional microplate reader (Molecular Devices, USA). A blank control without antibody and a negative control with the addition of an equal amount of Leptospira interrogans recombinant cytochrome C peroxidase (rLep-MauG) were set in the experiment.

And (3) ELISA detection results: rvWFA3-2 bound strongly to hCO OL1/4 but weakly to hCO OL3/6 as shown in FIG. 2.

7. Surface Plasmon Resonance (SPR) detection

hCOL1/3/4/6 was dissolved in 10mmol/L sodium acetate (pH4.5) to prepare a 100. mu.g/mL protein solution, which was immobilized on a CM5 chip by amino coupling. rvWFA3-2 was dissolved in mobile phase buffer (10 mmol/L HEPES, 150mmol/L NaCl, 3mmol/L EDTA, 0.05% P20, pH7.4) to a final concentration of 4, 8, 16, 32 or 64nmol/L, respectively. Binding conditions of rvWFA3-2 at different concentrations when flowing through a CM5 chip combined with hCOL1/3/4/6 were detected by a t200 BiaCore instrument (GE), and the obtained data were subjected to kinetics fitting analysis by Evaluation-3.0 software, and binding conditions between different proteins were reflected by equilibrium binding constants (KD). Blank controls without leptospira interrogans recombinant protein were set up in the experiment.

SPR detection result: rvWFA3-2 bound hCOL1/4 rapidly and remained in a stable bound state for a long time, and had KD values of 6.4X 10^-9And 3.2X 10^-9M, as in FIG. 3.

8. Real-time fluorescent quantitative RT-PCR (qRT-PCR) detection

The Primer Premier 5.0 software is adopted to design LA _4207 gene real-time fluorescent quantitative RT-PCR (qRT-PCR) primers, LA _4207 upstream primers: 5'-GAC GCT TCT GGG TCT A-3', downstream primer: 5'-CCA TTT CTA AAG GCT GA-3' are provided.Leptospira interrogans in EMJH medium were centrifuged and PBS washed as above and then counted under a dark field microscope using a Petroff-Hausser counting plate. 6 well cell culture plates were seeded 10 per well⁵HUVEC or EOMA cells, 2.5% FCS RPMI-1640 or DMEM medium at 37 ℃ for 12 h. HUVEC and EOMA cells are respectively infected for 1, 2, 4, 8, 12 or 24 hours according to the ratio of leptospira interrogans to cells which is 100:1, the cells are digested by 0.25 percent of pancreatin and then centrifuged for 15min at 1000r/min and 4 ℃, cell precipitates are removed, and the supernatant is centrifuged for 15min at 12000r/min and 4 ℃ to obtain extracellular leptospira interrogans precipitates. Extracting total RNA of leptospira interrogans by using a TRIzol bacterial RNA preparation kit (TaKaRa), removing residual DNA by using a gDNA removal kit (TaKaRa), and determining the RNA concentration by using an ultraviolet spectrophotometry. Using 1. mu.g of total RNA as a template, PrimeScript was added^TMIt was reverse-transcribed into cDNA with kit (TaKaRa), and then

The Premix Ex-TaqTM fluorescent quantitative PCR kit and a real-time fluorescent quantitative RT-PCR instrument (API, USA) detect the mRNA level of the LA _4207 gene, and the reaction parameters are as follows: 30s at 95 ℃; 5s at 95 ℃, 30s at 60 ℃ and 40 cycles. In the experiment, the Leptospira interrogans lysine strain 16S rRNA gene is used as an internal reference, and an upstream primer: 5'-CTT TCG TGC CTC AGC GTC AGT-3', downstream primer: 5'-CGC AGC CTG CAC TTG AAA CTA-3' are provided. Equal amounts of uninfected or Leptospira interrogans incubated for the same time in RPMI-1640 or DMEM medium were set as controls in the experiment, and qRT-PCR data were analyzed using the Delta Ct mathematical model and REST-2005 software.

Results of qRT-PCR: after infection of HUVEC and EOMA cells with Leptospira interrogans, vWFA3-2-mRNA levels increased significantly rapidly (P <0.05), and the transcriptional levels were unaffected by the different cell culture media, as shown in FIG. 4 (P <0.05 compared to the level of Lep4207-mRNA before infection). vWFA3-2 was postulated to act as an adhesion factor in leptospira infection.

9. Western Blot detection

Leptospira interrogans lys were used to infect HUVEC or EOMA cells for 1, 2, 4, 8, 12 or 24h as above and the extracellular Leptospira interrogans were pelleted by centrifugation. The Leptospira interrogans precipitate was suspended in sterile double-distilled water, sonicated in an ice bath, centrifuged at 12000r/min for 15min at 4 ℃, the supernatant was taken and ice-washed with 10% (W/V) trichloroacetic acid (TCA) (Sigma) for 60min to precipitate the protein, and the protein was washed twice with pre-cooled acetone to remove TCA. The protein precipitate was dissolved in sterile double distilled water and the protein concentration was determined by BCA method. A rabbit anti-rvWFA 3-2-IgG diluted at a ratio of 1:200 is taken as a primary antibody, a Horse Radish Peroxidase (HRP) labeled goat anti-rabbit IgG (Abcam) diluted at a ratio of 1:3000 is taken as a secondary antibody, a gene expression product in a sample is detected by a Western Blot method, and a positive hybridization strip is observed by a Bio-Rad gel imaging system.

Western Blot detection result: after Leptospira interrogans lys infect HUVEC and EOMA cells, vWFA3-2-mRNA level rapidly and remarkably rises (P <0.05) similar to qRT-PCR in a Western Blot detection result, and different cell culture media have no influence on the transcription level of the Leptospira interrogans lys, as shown in FIG. 5.

10. Immunological activity analysis of recombinant protein rvWFA3-2

Respectively adding 1: the 1000 diluted rabbit anti-leptospira whole serum and rvWFA3-2 antiserum were primary antibodies, 1: western blot analysis was performed using 5000-diluted horseradish peroxidase-labeled goat anti-rabbit IgG as a secondary antibody. The 96-well plate was coated with 1mg/ml of the purified recombinant protein rvWFA3-2, allowed to stand at 37 ℃ for 1h, and then blocked with 6% calf serum overnight at 4 ℃.

results of the immunological activity assay for rvWFA 3-2: a result of taking a rabbit anti-leptospira whole-bacterium serum and a rabbit anti-rvWFA 3-2 serum as primary anti-Western-blot respectively shows that a hybridization band appears at a corresponding position of the recombinant protein, and the recombinant protein expressed by escherichia coli is prompted to have immunological activity.

11. Minimum Lethality Determination (MLD)

Leptospira interrogans jaundice bleeding group lysine strain, influenza typhoid influenza cold type clinical 6 strain and Bomodona type Rostrain are measured for the 100% minimum fatality amount of Jindi. Inoculating golden hamster into abdominal cavity of the strain, collecting heart blood after infecting for 2-3 days, separating leptospira interrogans from 8% rabbit serum, so as to achieve the effect of enhancing the toxicity of the leptospira interrogans; ② freshly cultured jaundice bleeding group lysine strain, influenza typhoid influenza cold type limb 6 strain and Bomena RostrainCentrifuging at 12000r/min for 15min (4 ℃), resuspending the precipitated leptospira with EMJH culture solution with different volumes, counting by Petroff-Hausser counting chamber, and preparing leptospira suspension with different concentrations; ③ dividing 30 golden hamster into 5 groups, each group comprises 6 golden hamster, wherein 4 groups are respectively injected into 10 abdominal cavities⁷、10⁸、10⁹Or 10¹⁰1ml of leptospira interrogans lysine suspension, injecting an equal volume of EMJH culture solution into the other group as a control, and observing for 7 days under the conventional feeding condition; and fourthly, taking the lowest leptospira interrogans concentration of all the golden hamster dead in the observation period as the 100 percent lowest lethal dose (MLD) of the leptospira interrogans of the serogroup. The raising temperature of the experimental animals is 21 +/-2 ℃, the relative humidity is 30-7%, and the illumination period is 14/10 h.

MLD results: leptospira interrogans Treponema jaundice bleeding group lysine strain, influenza typhoid influenza cold type clinical 6 strain and Bomodona bolana type Rostrain are respectively 1.5 × 10 to 100% MLD of Jindi⁸、3.0×10⁹、1.5 ×10⁹。

12. rvWFA3-2 vaccine immunoprotection assay

200 ug of rvWFA3-2 was mixed with 1mg of aluminum hydroxide adjuvant to obtain rvWFA3-2 vaccine, 200 ug of BSA (Sigma) was mixed with 1mg of aluminum hydroxide as negative control, and rvWFA3-2 was mixed with aluminum hydroxide adjuvant and emulsified to obtain rvWFA3-2 vaccine. Male golden mice (purchased from beijing vindelia laboratory animal technology ltd) of 4 weeks of age were divided into 6 groups of 15 mice each. The protective effect of the rvWFA3-2 vaccine immunization on the main epidemic pathogenic leptospira interrogans hemorrhagic group lysine strain, influenza typhoid influenza cold type clinical 6 strain and Bomana Borna type Rous strain in China is detected. 1-3 groups inject rvWFA3-2 vaccine 0.1ml into one side groin subcutaneously, 4-6 groups inject negative control mixture 0.1ml into one side groin subcutaneously; ② repeating the immunization on the other side of the groin after 2 weeks; ③ 4 weeks after the last immunization,

groups

1 and 4 used 2-fold MLD Leptospira interrogans lysine (3.0X 10)⁸) 1ml suspension, 2 nd and 5 th groups with 2-fold MLD of influenza typhoid influenza type 6 strains (6.0X 10)⁹) 1ml of suspension, 2 times MLD of Bommona type Rous strain (3.0X 10) for groups 3 and 6⁹) 1ml of suspension; fourthly, the golden hamster is conventionally fedThe animals were observed for 7 days under nutrient conditions and the death status of each group was recorded. The raising temperature of the experimental animals is 21 +/-2 ℃, the relative humidity is 30-7%, and the illumination period is 14/10 h.

rvWFA3-2 vaccine immunoprotection Effect: results of the rvWFA3-2 vaccine golden hamster immunoprotection assay are shown in Table 1. The survival rates of the rvWFA3-2 vaccine immunized golden hamster after being attacked by 2 times MLD jaundice bleeding group lysine, influenza typhoid influenza type limb 6 and Bomana Borna type rib are 86.67%, 80.00% and 73.33% respectively, and the survival rates of the control group without rvWFA3-2 vaccine immunized golden hamster jaundice bleeding group lysine, influenza typhoid influenza type limb 6 and Bomana Borna type rib 2 times MLD are 6.67%, 0 and 6.67% respectively. The survival rate of the rvWFA3-2 vaccine immunized golden hamster after being attacked by pathogenic leptospira interrogans is obviously higher than that of a negative control group, the difference has statistical significance (P <0.05), and the rvWFA3-2 vaccine immunization has immunoprotection effect on the golden hamster.

TABLE 1 results of rvWFA3-2 vaccine immunization golden hamster immunoprotection test

Test group	Total number of animals (only)	Number of dead animals	Number of surviving animals (only)	Immunoprotection Rate (%)
					1	15	2	13	86.67
2	15	3	12	80.00
					3	15	4	11	73.33
4	15	14	1	6.67
					5	15	15	0	0
6	15	14	1	6.67

13. Microscopic Agglutination Test (MAT)

Blood separated serum from heart of the rvWFA3-2 vaccine immunized golden hamster which survived the animal protection test is collected. Serum was diluted with PBS at 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600, and 0.1ml of each was mixed with an equal amount of freshly cultured 9 groups of pathogenic leptospira 9 (leptospira icterohepatitis bleeding group lysine, influenza typhoid influenza type limb 6, autumn group autumn type limb 4, pomona type, australia type 65-9, seven day fever type 56069, canine type forest, and serogroup Ulva type L183), incubated at 37 ℃ for L h, and then observed under a dark field microscope, and the highest dilution of 50% of the leptospira agglutinated serum was used as the titer determination endpoint. Normal golden hamster serum was used as a control in the experiment.

MAT results: as shown in Table 2, the obtained immune-surviving golden hamster serum antibody can agglutinate with 9 groups of 9 type Leptospira interrogans strains, and the MAT serum antibody titer is 1:50-1:800, which indicates that the rvWFA3-2 immunity can generate titer protective antibodies and has cross protection effect.

TABLE 2 rvWFA3-2 vaccine immunization golden hamster serum MAT detection result

LA _4207 (52-627) gene fragment amplification primer:

an upstream primer: 5' -CGCCAT ATG(Nde I)GTAACTGGAACAAACGTATCTTCA-3’，

A downstream primer: 5' -CGCCTC GAG(Xho I)CCTAAGCGAATCGAG AGCGGTATA-3’

Nucleotide sequence (1) … (576) of gene fragment of lysine strain LA _4207 (52-627) of icterohaemorrhagic hemorrhage group, amino acid sequence (1) … (192) of vWFA3-2 protein

(1)

GAA AAT CAA ACG CCA TCC CAA TTG

(1)

Glu Asn Gln Thr Pro Ser Gln Leu

(49)TTT ATC ATA GAC GCT TCT GGG TCT ATG AAC GAA TAC TTA GGG ATT TAT

(17)Phe Ile Ile Asp Ala Ser Gly Ser Met Asn Glu Tyr Leu Gly Ile Tyr

(97)CAA AAA ATT CAT TTA GCT AAG AAA CAC GTT AGT CAC TAT ATA TCC ACT

(33)Gln Lys Ile His Leu Ala Lys Lys His Val Ser His Tyr Ile Ser Thr

(145)CTT CCT CAA GAA ACT GAA ATC GGT TTT TTA GCC TAT GGA AAT CGG CTG

(49)Leu Pro Gln Glu Thr Glu Ile Gly Phe Leu Ala Tyr Gly Asn Arg Leu

(193)CCA GGA TGT TCG TCC TCA AGA TTG TAT CAG CCT TTA GAA ATG GGA AAT

(65)Pro Gly Cys Ser Ser Ser Arg Leu Tyr Gln Pro Leu Glu Met Gly Asn

(241)CGC GAT ACT TTT AAA AAC CGA CTA TTT AGT CTA ACT CCT TCG GGT GCT

(81)Arg Asp Thr Phe Lys Asn Arg Leu Phe Ser Leu Thr Pro Ser Gly Ala

(289)ACA CCT CTC GCA GAA TCA ATT CGA ATC GCA GGA ACT TTA ATT TCG CAA

(97)Thr Pro Leu Ala Glu Ser Ile Arg Ile Ala Gly Thr Leu Ile Ser Gln

(337)AGA AAA AAG GAA ACT GAA ATT ATT TTA GTG ACG GAC GGT ATA GAA AGT

(113)Arg Lys Lys Glu Thr Glu Ile Ile Leu Val Thr Asp Gly Ile Glu Ser

(385)TGT TAC GGA GAT CCT AAA AAA GAA CTT CAA GCA CTC AAA CAA AAA GGA

(129)Cys Tyr Gly Asp Pro Lys Lys Glu Leu Gln Ala Leu Lys Gln Lys Gly

(433)ATC CCA TTT CAA TTT CAT GTC CTC GGT CTC GGG CTC AAA AGT CAT GAG

(145)Ile Pro Phe Gln Phe His Val Leu Gly Leu Gly Leu Lys Ser His Glu

(481)GAG TTA CAA ATG AAA ATT CTC ACA GAG GAA GGA AAT GGA AAG TAT TTC

(161)Glu Leu Gln Met Lys Ile Leu Thr Glu Glu Gly Asn Gly Lys Tyr Phe

(529)AGT ATC GAG GAC GAT TCT TCT TTT

(177)Ser Ile Glu Asp Asp Ser Ser Phe

Note: □, primer sequences; the LA _0697 gene superfamily domain sequence is underlined.

Sequence listing

<110> Hangzhou college of medicine

<120> preparation method and application of leptospira adhesion protein rvWFA3-2

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 33

<212> DNA

<213> Forward primer

<400> 1

cgccatatgg taactggaac aaacgtatct tca 33

<210> 2

<211> 33

<212> DNA

<213> downstream primer (downstream primer)

<400> 2

cgcctcgagc ctaagcgaat cgagagcggt ata 33

<210> 3

<211> 576

<212> DNA

<213> nucleotide sequence of rvWFA3-2 (rvWFA3-2 nucleotide sequence)

<400> 3

gtaactggaa caaacgtatc ttcagaaaat caaacgccat cccaattgtt tatcatagac 60

gcttctgggt ctatgaacga atacttaggg atttatcaaa aaattcattt agctaagaaa 120

cacgttagtc actatatatc cactcttcct caagaaactg aaatcggttt tttagcctat 180

ggaaatcggc tgccaggatg ttcgtcctca agattgtatc agcctttaga aatgggaaat 240

cgcgatactt ttaaaaaccg actatttagt ctaactcctt cgggtgctac acctctcgca 300

gaatcaattc gaatcgcagg aactttaatt tcgcaaagaa aaaaggaaac tgaaattatt 360

ttagtgacgg acggtataga aagttgttac ggagatccta aaaaagaact tcaagcactc 420

aaacaaaaag gaatcccatt tcaatttcat gtcctcggtc tcgggctcaa aagtcatgag 480

gagttacaaa tgaaaattct cacagaggaa ggaaatggaa agtatttcag tatcgaggac 540

gattcttctt tttataccgc tctcgattcg cttagg 576

<210> 4

<211> 192

<212> PRT

<213> rvWFA3-2 amino acid sequence (rvWFA3-2 amino acid sequence)

<400> 4

Val Thr Gly Thr Asn Val Ser Ser Glu Asn Gln Thr Pro Ser Gln Leu

1 5 10 15

Phe Ile Ile Asp Ala Ser Gly Ser Met Asn Glu Tyr Leu Gly Ile Tyr

20 25 30

Gln Lys Ile His Leu Ala Lys Lys His Val Ser His Tyr Ile Ser Thr

35 40 45

Leu Pro Gln Glu Thr Glu Ile Gly Phe Leu Ala Tyr Gly Asn Arg Leu

50 55 60

Pro Gly Cys Ser Ser Ser Arg Leu Tyr Gln Pro Leu Glu Met Gly Asn

65 70 75 80

Arg Asp Thr Phe Lys Asn Arg Leu Phe Ser Leu Thr Pro Ser Gly Ala

85 90 95

Thr Pro Leu Ala Glu Ser Ile Arg Ile Ala Gly Thr Leu Ile Ser Gln

100 105 110

Arg Lys Lys Glu Thr Glu Ile Ile Leu Val Thr Asp Gly Ile Glu Ser

115 120 125

Cys Tyr Gly Asp Pro Lys Lys Glu Leu Gln Ala Leu Lys Gln Lys Gly

130 135 140

Ile Pro Phe Gln Phe His Val Leu Gly Leu Gly Leu Lys Ser His Glu

145 150 155 160

Glu Leu Gln Met Lys Ile Leu Thr Glu Glu Gly Asn Gly Lys Tyr Phe

165 170 175

Ser Ile Glu Asp Asp Ser Ser Phe Tyr Thr Ala Leu Asp Ser Leu Arg

180 185 190

Claims

1. A preparation method of leptospira adhesion protein rvWFA3-2 is characterized by comprising the following steps:

(1) designing PCR primers containing endonuclease Nde I or Xho I enzyme cutting sites according to restriction endonuclease map analysis and signal peptide prediction results of a T-A cloning and subcloning vector pET42a multiple cloning sites of a Leptospira interrogans lysine strain LA _4207 gene, and reserving for later use, wherein the nucleotide sequence of an upstream primer in the PCR primers is shown as SEQ ID No.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 2;

(2) extracting Leptospira interrogans lysine strain DNA, centrifuging a Leptospira interrogans lysine strain culture at 12000r/min and 4 ℃ for 15min to obtain a precipitate, washing with sterilized PBS (0.01 mol/L and pH7.2), centrifuging according to the method, repeating for 2 times to obtain a Leptospira interrogans precipitate, and extracting DNA by using a bacterial genome DNA preparation kit;

(3) amplifying a superfamily structural domain fragment of a coagulation factor A3 area in the LA _4207 gene by adopting the PCR primer obtained in the step (1) and the Leptospira interrogans lysine strain DNA obtained in the step (2) to obtain a target gene amplification fragment, detecting the amplification fragment by ethidium bromide pre-stained 1.5% agarose gel electrophoresis, and then connecting the target gene amplification fragment with pMD19-T by adopting a T-A cloning kit, wherein the amplification method comprises the following steps: carrying out amplification reaction by using EX-Taq high-fidelity PCR kit with PCR parameters of 94 ℃ for 5min, 94 ℃ for 30s, 50 ℃ for 30s, 72 ℃ for 90s, 30 cycles and 72 ℃ for 7min, connecting overnight, and transforming the plasmid into competent E.coli DH5 alpha by using a calcium chloride method to form E.coli DH5 alpha^pMD19-T4207Extracting recombinant plasmid pMD19-T after screening blue and white⁴²⁰⁷Sequencing;

(5) pET42a is prepared by calcium chloride method⁴²⁰⁷Transformation into competent E.coli BL21DE3 to form E.coli BL21DE3^pET42a-4207Engineering strain, inoculating to 50 μ g/mL kanamycin LB agar plate for isolated culture, inducing with 0.5mmol/L IPTG to obtain target recombinant protein rvWFA3-2, purifying rvWFA3-2 with Ni-NTA affinity chromatographic column; the separation culture and IPTG induction method comprises the following steps: shake culturing at 37 deg.C at 220r/min to A₆₀₀When the value is 0.6-0.8, 0.5mmol/L IPTG is added, and the culture is continued for 6h according to the method, so as to induce the expression of the target recombinant protein rvWFA 3-2.

2. The application of the leptospira adhesion protein rvWFA3-2 in preparing vaccines for treating leptospira interrogans infection is disclosed, wherein the amino acid sequence of the leptospira adhesion protein rvWFA3-2 is shown as SEQ ID No. 4.

3. The application of the leptospira adhesion protein rvWFA3-2 in the preparation of a leptospira infection antibody detection kit is disclosed, wherein the amino acid sequence of the leptospira adhesion protein rvWFA3-2 is shown as SEQ ID No. 4.