CN108300682A

CN108300682A - A kind of haemophilus parasuis OmpP2 gene-deleted strains and its construction method and application

Info

Publication number: CN108300682A
Application number: CN201810127495.2A
Authority: CN
Inventors: 于江; 吴家强; 王金宝; 杨杰; 张玉玉; 李建达; 曾昊
Original assignee: Institute Animal Science and Veterinary Medicine of Shandong AAS
Current assignee: Institute Animal Science and Veterinary Medicine of Shandong AAS
Priority date: 2017-11-13
Filing date: 2018-02-08
Publication date: 2018-07-20

Abstract

The present invention relates to gene engineering technology fields, and in particular to a kind of haemophilus parasuis OmpP2 gene-deleted strains, deposit number：CCTCC NO：M 2017574, after gene delection, the gene-deleted strain of acquisition is relative to haemophilus parasuis LC strain, virulence reduces, and the speed of growth slows down, the pathogenic reduction to mouse, adherency and invasive ability to host cell weaken, and have broad application prospects in terms of studying haemophilus parasuis molecular mechanism.

Description

A kind of haemophilus parasuis OmpP2 gene-deleted strains and its construction method and application

Technical field

The present invention relates to gene engineering technology fields, and in particular to a kind of haemophilus parasuis OmpP2 gene-deleted strains, also It is related to construction method and the application of this haemophilus parasuis OmpP2 gene-deleted strains.

Background technology

Haemophilus parasuis (Haemophilus parasuis, HPS) is under the jurisdiction of Pasteurellaceae (Pasteurellaceae) hemophilus, is a kind of polymorphic dialister bacterium, the bacterium not haemolysis, and does not have motility. Microscopically observation, the bacterium are Gram-negative bacterias, and form differs, from single coccobacillus to long, elongated, so that it is Filamentous Thalline.HPS often causes polyserositis, arthritis and the meningitis of pig, and pig is also known as by the microbial diseaseDisease.The bacterium is separated in nineteen twenty-two by Schermer and Ehrlich for the first time, but is just passed through until 1992 SPF (Specific Pathogen Free, no-special pathogen) pig confirm HPS pathogenic and different serotypes bacterial strain it Between Virulence Difference.HPS can influence the monthly age pig of 2 week old~4, and mainly wean is front and back and the piglet in child care stage, usually See the pig of the week old of 5 week old~8, incidence is up to 40%, and the death rate is up to 50% when serious.

In recent years, intensive intensive cultivation, the unordered influence introduced a fine variety made the arthritis caused by HPS, pericarditis, peritonaeum Scorching report is more and more, especially some inhibitive ability of immunity diseases such as porcine reproductive and respiratory syndrome, Porcine circovirus desease etc. Often with HPS mixed infections or secondary infection, huge economic loss is caused to the pig breeding industry in the whole world, also gives haemophilus parasuis The diagnosis and integrated control of disease bring huge difficulty.

It is more and more about the virulence gene or pathogenicity island of HPS and its pathogenic mechanism research, it has been determined that HPS virulence factors Include mainly lipopolysaccharides, turn iron-binding protein, neuraminidase, capsular polysaccharide, outer membrane protein, hemolysin operon hhdBA It is found have certain correlation with virulence, but still needs to further study confirmation.

Outer membrane protein (Outer membrane protein, Omp) is the peculiar structure of Gram-negative bacteria, in cell wall More than half is accounted in component content, has the function of sertoli cell structure, transport nutriment, in conjunction with phagocytosis receptor body etc..Have Research shows that Omp is related with the virulence of HPS, and variant from sodium selenite and the Omp that is separated in illness pig, from The Omp detached in the sick pig body of polyserositis and arthritic symptom is essentially identical.

The virulence of HPS OmpP2 gene pairs host cells and its pathogenic effects in host cell are still not clear, therefore, It is very necessary to obtain a kind of HPS of OmpP2 gene delections.CN103865834A discloses a kind of missing 394131- The dual-gene gene-deleted strain of 181258-181643 nucleotide of 394631 nucleotide and omp2 gene delections, structure Hps is without anti- Property label dual-gene deletion mycopremna, and its biological characteristics and pathogenicity etc. are inquired into, are that further development of new is high The genetic engineering live vaccine that effect can provide high cross protection effect lays the first stone.But in this patent still without clear OmpP2 The direct relation of gene and virulence.

Invention content

In order to solve the virulence of HPS OmpP2 gene pairs host cells and its asking for the pathogenic effects in host cell Topic, the present invention constructs one plant of HPS gene-deleted strain, using homologous recombination technique, is struck on the basis of LC plants of 1 types of HPS It removes OmpP2 portion genes and obtains, which send on October 11st, 2017 into China typical culture collection Heart preservation is named as haemophilus parasuis LC Δs OmpP2 (Haemophilus parasuis LC Δ OmpP2), deposit number： CCTCC NO：M 2017574, address：Wuhan, China Wuhan University.

The haemophilus parasuis OmpP2 gene-deleted strain LC Δ OmpP2 are lacked relative to haemophilus parasuis LC strain The sequence of mistake is shown in sequence 12 in sequence table.

The haemophilus parasuis OmpP2 gene-deleted strain LC Δ OmpP2, relative to haemophilus parasuis LC strain, poison Power reduces.

A kind of construction method of haemophilus parasuis OmpP2 gene-deleted strains, includes the following steps：

(1) the upper and lower homology arm of OmpP2 genes is expanded, genetic fragment OmpP2up and OmpP2down are obtained；

(2) gentamicin resistance gene is expanded, genetic fragment Gm is obtained；

(3) genetic fragment OmpP2up is connect to obtain OmpP2up-Gm with Gm, then connect with OmpP2down to obtain gene Segment OmpP2up-down；

(4) genetic fragment OmpP2up-down is connected in suicide plasmid pK18mobsacB by digestion, obtains weight Group plasmid pK18-OmpP2；

(5) recombinant plasmid transformed haemophilus parasuis to get.

Using HPS LC pnca genes groups as template, with OmpP2up-F and OmpP2up-R, OmpP2down-F and OmpP2 Down-R is primer, carries out PCR amplification, amplified band shows that size is about 500bp or so through electrophoretogram, with expected purpose Segment 478bp is consistent with 525bp, and amplified production is recycled.Since HPS is to gentamicin (Gm) sensitivity, use primer from matter Gentamicin resistance gene is expanded on grain so that as selection markers, amplified production is shown through electrophoretogram, band is about the left sides 750bp The right side, size is consistent with expected purpose segment 783bp, and amplified production is recycled.By OmpP2up, OmpP2down and Gm piece of recycling Section is connected by Overlap PCR, and amplified production is recycled.Use BamHI and HindIII that will recycle segment and suicide plasmid respectively PK18mobsacB carries out double digestion.Target fragment recycles, 16 DEG C of connections overnight, connection product Transformed E .coli DH5 α.By Gm The positive clone of resistant gene PCR identifications expands culture, extracts plasmid, is identified with BamHI and HindIII double digestions.Double digestion Electrophoretogram shows two bands, and a band is more than 5000bp, and another stripe size is about 1800bp, with expected 1749bp purposes Segment is consistent with pK18mobsacB plasmid sizes, will identify that correct recombinant plasmid is named as pK18-OmpP2.By recombinant plasmid PK18-OmpP2 Natural Transformations enter in LC plants, with the TSA tablets of Gm (25 μ g/ml) resistance (containing 5% serum and 0.001%NAD) Screen transformant.Monoclonal in picking resistant panel to containing Gm (25 μ g/ml) TSB culture mediums (containing 5% serum and In 0.001%NAD), it is incubated overnight.Using Gm-F, Gm-R as primer, gentamicin resistance gene is expanded, is obtained more than 500bp's Gm genetic fragments, amplification show that Gm segments are successively inserted into genome.Design pair of primers OmpP2-1, OmpP2-2 amplification The OmpP2 segments of missing, it is that template carries out pcr amplification reaction, no amplified band that positive transformant, which is incubated overnight bacterium solution,.With LC plants As a contrast, pcr amplification reaction obtains the band of 1194bp.PCR product electrophoretogram, which is shown, has successfully lacked OmpP2 genes Overall length.By the LZ screened strain OmpP2 gene-deleted strains on the TSA tablets containing Gm blind passage, with primer Gm-F, Gm-R and OmpP2-1, OmpP2-2 identify every generation qualification result shows that continuously passing for 5 generations does not all amplify OmpP2 bases Because of segment.

The primer sequence of the construction method, the middle amplification upper and lower homology arm of OmpP2 genes of step (1) is as follows：

OmpP2up-F：5'-ATAGGATCCACCGCTTGTTCTCTGTATTAAGCGTATAG-3',

OmpP2up-R：5'-GTTGCTGCTGCGTAACATAATGTTCACCTTAAGTTTTT-3',

OmpP2down-F：5'-CCAAGTACCGCCACCTAATCTAGATAGTTAGGTACTAT-3',

OmpP2down-R：5'-TTAAAGCTTACAAGCGGTGCTGAACAGGGTAAAGTAGC-3'。

The primer sequence of the construction method, the middle amplification Gm of step (2) is as follows：

Gm-F:5'-ATGTTACGCAGCAGCAACGATG-3',

Gm-R:5'-TTAGGTGGCGGTACTTGGGT-3'。

The haemophilus parasuis OmpP2 gene-deleted strains LC Δs OmpP2 is in host cell is tested and is pathogenic Using.

The principle of the present invention is the homologous recombination technique of gene, passes through the homologous sequence between exogenous DNA and chromosomal DNA It recombinates.We realize homologous recombination by the method for Natural Transformation.Natural Transformation method is the intake of bacterium competent cell Exogenous DNA and the method for being stablized heredity.The foundation of haemophilus parasuis competent cell is by cAMP receptor protein CRP tune Control, the raising of cAMP concentration can activate its receptor protein CRP.

Compared with prior art, the present invention has the advantages that following prominent：

1. the material used in the present invention is wild pathogenic strain LC plants of HPS, which is isolated from morbid pig One plant of serum 1 type bacterial strain, have to pig it is stronger pathogenic, can not only provide it is homologous attack malicious protection, can also be 4 type of serum, 5 types, The heterologous poison of attacking such as 10 types, 12 types, 14 types and 15 type HPS provides certain cross protection.Therefore, it is further built with the pathogenic bacteria OmpP2 gene-deleted strains have broad application prospects in terms of studying HPS molecular mechanisms；

After 2.HPS OmpP2 gene delections, the gene-deleted strain speed of growth of acquisition slows down, the pathogenic reduction to mouse；

After 3.HPS OmpP2 gene delections, the gene-deleted strain of acquisition to the adherency of host cell (porcine alveolar macrophage) and Invasive ability weakens；

The construction method of 4.HPS OmpP2 gene-deleted strains is also applied for the structure of other gene-deleted strains of HPS；

5.HPS OmpP2 gene-deleted strain LC Δs OmpP2 can be used as HPS OmpP2 gene functional research and pathogenic mechanism The reference strain of research.

Description of the drawings

Fig. 1 OmpP2up and OmpP2down PCR amplification are as a result, M：DL2000；1-6：OmpP2up amplifications；7-11： OmpP2 down amplifications；

Fig. 2 Gm gene PCR amplifications, M：DL2000；1-2：Gm amplifications；

Fig. 3 recombination suicide vector digestion qualification results；

Fig. 4 recombinant plasmid pK18-OmpP2 collection of illustrative plates；

Fig. 5 HPS gene-deleted strain PCR qualification results, A：Primer HPS-F/R amplifications；B：Primer Gm-F/R amplifications；C：Primer OmpP2-1/2 is expanded；D：Primer OmpP2up-F/down-R expands M：DL2000/DL5000；1-4：HPS gene-deleted strains；LC： LC plants of HPS；

The relationship of Fig. 6 HPS wild strains and gene-deleted strain growth time and OD600nm；

Fig. 7 HPS wild strains adhere to porcine alveolar macrophage test result with gene-deleted strain；

Fig. 8 HPS wild strains invade porcine alveolar macrophage test result with gene-deleted strain.

Culture presevation information

The preservation time：On October 11st, 2017,

Depositary institution：China typical culture collection center,

Deposit number：CCTCC NO：M 2017574,

Depositary institution address：Wuhan, China Wuhan University, postcode：430072

Classification And Nomenclature：Haemophilus parasuis Haemophilus parasuis.

Specific implementation mode

With reference to specific embodiment, invention is further explained：

Embodiment 1

The amplification of the upper and lower homology arm of 1.HPS OmpP2 genes

According to OmpP2 gene orders (FJ685759.1) design primer for the HPS H49 bacterial strains announced on GenBank, with HPS serum 1 type LC pnca gene groups are template, expand the upstream and downstream homology arm of OmpP2 genes.In upstream, the upstream of homology arm is drawn BamHI restriction enzyme sites and USS sequences, the downstream primer OmpP2 down of homology arm in downstream is added in the ends object OmpP2up 5 '- 5 '-ends introduce HindIII restriction enzyme sites and USS sequences.The primer sequence is as follows：

OmpP2up-F：5'-ATAGGATCCACCGCTTGTTCTCTGTATTAAGCGTATAG-3'

(underscore represents BamHI restriction enzyme sites；Italic represents USS sequences)

OmpP2up-R：5'-GTTGCTGCTGCGTAACATAATGTTCACCTTAAGTTTTT-3'

Primer OmpP2up-F and OmpP2up-R expand upstream homology arm 480bp.

OmpP2down-F：5'-CCAAGTACCGCCACCTAATCTAGATAGTTAGGTACTAT-3'

OmpP2down-R：5'-TTAAAGCTTACAAGCGGTGCTGAACAGGGTAAAGTAGC-3'

(underscore represents HindIII restriction enzyme sites；Italic represents USS sequences)

Primer OmpP2down-F and OmpP2down-R expand downstream homology arm 528bp.

By LC plants of the HPS serum 1 types of freeze-drying in TSA tablets (tryptose soya agar culture medium, purchased from U.S. BD public affairs Department, addition volume are 5% newborn bovine serum and 0.001% coenzyme NAD) on recover after 2 generations, picking single bacterium colony is placed in 100 μ In L aqua sterilisas, after blowing and beating mixing, boil 10min, multigelation twice after, 5000rpm centrifuges 5min, takes supernatant as template, PCR amplification system is 50 μ l, and reaction system is as follows：10 × PCR buffer, 5 μ L, 2.5mmol/L dNTP 4 μ L, MgCl₂ 3μ L, 2 μ L of sense primer, 2 μ L, TaqDNA Polymerase of downstream primer 1 μ L, template 4 μ L, ddH₂O is supplemented to 50 μ L.

PCR amplification condition is：95 DEG C of pre-degenerations 5min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 35 cycles, 72 DEG C are prolonged Stretch 10min.Amplified band is analyzed through 1% agargel electrophoresis, about 500bp or so, with expected target fragment 478bp and 525bp is consistent (Fig. 1), and amplified production is recycled, and purifies target fragment.

2. the amplification of gentamicin resistance gene (Gm)

Select gentamicin as selection markers, with reference to the Gm resistance gene sequences on pFastBacI plasmids, design is available In the primer amplification Gm genes of Overlap PCR amplifications.Primer sequence is as follows：

Gm-F:5'-ATGTTACGCAGCAGCAACGATG-3'

Gm-R:5'-TTAGGTGGCGGTACTTGGGT-3'

Using pFastBacI plasmids as template, reaction system is as follows：10 × PCR buffer, 5 μ L, 2.5mmol/L dNTP 4 μ L, MgCl₂3 μ L, 2 μ L of sense primer, 2 μ L, TaqDNA Polymerase of downstream primer 1 μ L, template 4 μ L, ddH₂O is supplemented To 50 μ L.

PCR amplification condition is：95 DEG C of pre-degenerations 5min, 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 35 cycles, 72 DEG C are prolonged Stretch 10min.Amplified band is analyzed through 1% agargel electrophoresis, about 7500bp or so, with expected target fragment 783bp Unanimously (Fig. 2), amplified production is recycled, and purifies target fragment.

3.Overlap PCR

The OmpP2 up of recovery purifying are connect with Gm by Overlap PCR, reaction system is as follows：10×PCR 10 μ L, 2.5mmol/L dNTP of buffer 5 μ L, sense primer OmpP2up-F 2 μ L, downstream primer Gm-R 2 μ L, FastPfu 1 μ L of DNA Polymerase, template OmpP2up, Gm each 2 μ L, ddH₂O is supplemented to 50 μ L.PCR amplification condition is：95 DEG C of pre- changes Property 5min, 95 DEG C of 45s, 60 DEG C of 1min, 72 DEG C of 1min, 35 cycle, 72 DEG C extension 10min.Amplified band is through 1% agar Gel electrophoresis analysis, about 1200bp or so, it is consistent with expected target fragment 1242bp, amplified production is recycled, is purified Target fragment OmpP2up-Gm.

The OmpP2up-Gm of recovery purifying is connect with OmpP2down by Overlap PCR, reaction system is same as above.PCR Amplification condition is：95 DEG C of pre-degenerations 5min, 95 DEG C of 1min, 60 DEG C of 1min, 72 DEG C of 1min30s, 35 cycles, 72 DEG C of extensions 10min.Amplified band is analyzed through 1% agargel electrophoresis, occurs being more than 1500bp or so, with expected target fragment 1749bp Unanimously, amplified production is recycled, purifying target fragment OmpP2up-down.

4. building recombination suicide vector pK18-OmpP2

Use BamHI and HindIII that the OmpP2up-down of suicide plasmid pK18mobsacB and recovery purifying is bis- respectively Digestion.Target fragment recycles, and 16 DEG C of water-baths connect overnight, connection product Transformed E .coli DH5 α.By Gm resistant genes PCR The positive clone of identification expands culture, extracts plasmid, is identified with BamHI and HindIII double digestions.Double digestion electrophoretogram is shown Two bands, a band be more than 5000bp, another stripe size is about 1800bp, with expected 1749bp target fragments and PK18mobsacB plasmid sizes are consistent (Fig. 3), will identify that correct recombinant plasmid is named as pK18-OmpP2 (Fig. 4).5. recombination LC plants of plasmid Natural Transformation HPS

From the intensive scribing line of 3 single bacterium colonies of picking on the LC tablets of activation and the TSA tablets (new born bovine that addition volume is 5% Serum and 0.001% coenzyme NAD), 37 DEG C of 12~16h of incubator culture.With TSB fluid nutrient mediums, (addition volume is 5% Newborn bovine serum and 0.001% coenzyme NAD) wash lower lawn, after 10 times of dilutions, adjust OD_600nm=0.6.Take 20 μ L bacterium solutions in In 1.5mL sterile centrifugation tubes, 20 μM of cAMP is added, 10min is stood after mixing；2~5 μ g recombination suicide vectors are added PK18-OmpP2, gently mixing.Above-mentioned solution is transferred to TSA tablets (the newborn ox blood that addition volume is 5% of diameter 70mm Cleer and peaceful 0.001% coenzyme NAD) on, 37 DEG C are just set culture 6h.With 400 μ L TSB fluid nutrient mediums, (addition volume is 5% Newborn bovine serum and 0.001% coenzyme NAD) wash lower culture, take 50 μ L to be coated on the TSA tablets of Gm (25 μ g/mL) resistance (addition volume is 5% newborn bovine serum and 0.001% coenzyme NAD), 37 DEG C of cultures are to growing transformant.

The identification of LC plants of OmpP2 gene-deleted strains of 6.HPS

The monoclonal transformant grown after picking Natural Transformation (contains 5% blood to the TSB culture mediums containing Gm (25 μ g/ml) Cleer and peaceful 0.001%NAD) in, it is incubated overnight, is expanded with primer HPS-F, HPS-R, obtain the band (A in Fig. 5) of 821bp, explanation Transformant is HPS.It is expanded with primer Gm-F, Gm-R, obtains the gentamicin resistance gene (B in Fig. 5) of about 750bp, with drawing The OmpP2 segments of object OmpP2-1, OmpP2-2 amplification missing, no amplified band, as a contrast with LC plants, pcr amplification reaction obtains To the band (C in Fig. 5) of 1194bp.It is expanded with primer OmpP2up-F, OmpP2down-R, obtains the band of 1700bp or so, And the band (D in Fig. 5) more than 2000bp is arrived in LC plants of amplifications.PCR product electrophoretogram, which is shown, has successfully lacked OmpP2 genes Overall length.

HPS-F：5 '-ACCAGAAGCAAACCCAGAGC-3 ',

HPS-R：5 '-ACACCGCTTGCCATACCCTC-3 ',

OmpP2-1：5 '-ATGAAAAAAACACTAGTAGCATTAGC-3 ',

OmpP2-2：5’-TTACCATAATACACGTAAACCAACA-3’.

Sequence 11 in sequence table are with primer OmpP2up-F and OmpP2down-R amplification OmpP2 gene-deleted strain LC Δs

The sequence of OmpP2.

7.HPS gene-deleted strains growth time and OD_600nmThe measurement of relationship

LC plants of the HPS OmpP2 gene-deleted strains being incubated overnight and wild strain are transferred to 10mL TSB in 1% ratio In culture medium (containing 5% serum and 0.001%NAD), 37 DEG C of 180rpm shaken cultivations measure the bacterium solution taken out per hour OD_600nm, determine growth time and OD_600nmRelationship (Fig. 6), as a result, it has been found that the gene-deleted strain speed of growth is slower than wild strain.

The mouse challenge test of 8.HPS OmpP2 gene-deleted strains

The HPS gene-deleted strains of the invention of picking 2~3 and control LC plants of monoclonals of parent plant are intensive respectively to line The culture (containing 5% serum and 0.001%NAD) on TSA culture mediums, it is 10 to be adjusted to viable count⁷、10⁸、10⁹After CFU/mL, respectively 6 week old female Balb/c mouse of intraperitoneal inoculation, every group 10, only, the identical approach same dose of control group injects PBS to 0.2mL/. 12h starts to fall ill after generally connecing poison, is an observation period to meet 7d after poison, the death toll of each group mouse is counted, by Reed- MuenchShi methods calculate LD₅₀.Table 1 is statistical data.Calculation formula is：

lgLD₅₀=distance is higher than the logarithm of the dilution of 50% lesion rate than × difference between dilution logarithm+

1 mouse challenge test death toll statistical data of table

It is calculated according to formula, the LD of HPS wild strains₅₀It is 10^8.5The LD of CFU/mL, HPS gene-deleted strain₅₀It is 10^9.09CFU/mL。 The HPS gene-deleted strains virulence of the present invention declines compared with wild strain.

Application of the 9.HPS OmpP2 gene-deleted strains in host cell

The preparation of 9.1HPS wild strains and gene-deleted strain bacterium solution

Respectively by HPS OmpP2 gene-deleted strains and LC plant of wild strain it is intensive line TSA tablets (contain 5% serum and 0.001%NAD), 37 DEG C of culture 12h, are rinsed with 1640 sterile culture mediums respectively, and it is 10 to be diluted to viable count⁷CFU/mL。 9.2 adherence test

10⁵Porcine alveolar macrophage access 24 orifice plates, 37 DEG C of CO₂Culture solution, sterile PBS is sucked out in incubator overnight incubation It rinses twice, is respectively connected to LC plants of HPS OmpP2 gene-deleted strains and wild strain, the holes 1mL/, it is sterile that ghost control accesses 1mL 1640 culture mediums, 37 DEG C of CO₂The digestion of 200 μ L pancreatin, 37 DEG C of effects are added in incubator culture 3h, sterile PBS flushings 5 times 5min is added the deionized water of 800 μ L coolings, scrapes cell, repeatedly pressure-vaccum, draw 100 μ L and be serially diluted with deionized water, applied Plate, 37 DEG C of 24~48h of growth, experiment is in triplicate.The bacterium number grown is intracellular bacterium number, bacterial population=total bacterium of adherency Number-intracellular bacterium number.The bacterial population of LC plants of adherency of HPS wild strains is 1.5 × 10⁶CFU/mL, HPS OmpP2 gene-deleted strains adhere to Bacterial population be 1.0 × 10⁶CFU/mL (Fig. 7).

9.2 invasion experiments

10⁵Porcine alveolar macrophage access 24 orifice plates, 37 DEG C of CO₂Culture solution, sterile PBS is sucked out in incubator overnight incubation It rinses twice, is respectively connected to LC plants of HPS OmpP2 gene-deleted strains and wild strain, the holes 1mL/, it is sterile that ghost control accesses 1mL 1640 culture mediums, 37 DEG C of CO₂Incubator culture 3h, sterile PBS are acutely rinsed 5 times, and culture medium (the 5 μ g/ containing benzyl penicillin are added 100 μ g/mL of mL and gentamicin), 37 DEG C of CO₂Incubator culture 3h kills extracellular and cell surface adhesion HPS, sterile PBS is flushed three times, and the digestion of 200 μ L pancreatin is added, and 37 DEG C of effect 5min are added the deionized water of 800 μ L coolings, scrape cell, Pressure-vaccum repeatedly is drawn 100 μ L and is serially diluted with deionized water, coated plate, 37 DEG C of 24~48h of growth, count of bacteria on TSA tablets, Experiment is in triplicate.The bacterial population of LC plants of invasions of HPS wild strains is 9.2 × 10⁴CFU/mL, HPS OmpP2 gene-deleted strains enter The bacterial population invaded is 4.0 × 10⁴CFU/mL (Fig. 8).

By the mouse challenge test of above-mentioned HPS OmpP2 gene-deleted strains, it is found that HPS gene-deleted strain virulence is wilder Raw strain declines, and HPS OmpP2 gene-deleted strains are tested host cell attachment and invaded and test, after showing OmpP2 gene delections, HPS is adhered to and the ability of invasion host cell is obviously reduced, and illustrates that the virulence of OmpP2 gene pairs HPS has a great impact.Cause This, HPS OmpP2 gene-deleted strain LC Δs OmpP2 can be used for HPS OmpP2 gene functional research and pathogenic mechanism research, Have broad application prospects in terms of studying HPS molecular mechanisms.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not limited by embodiment System, other any changes made without departing from the spirit and principles of the present invention, modification, combination, replacement, simplification should be Equivalence replacement mode, is included within the scope of the present invention.

Sequence table

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gcggcttacg ttctgcccaa gtttgagcag ccgcgtagtg agatctatat ctatgatctc 960

gcagtctccg gcgagcaccg gaggcagggc attgccaccg cgctcatcaa tctcctcaag 1020

catgaggcca acgcgcttgg tgcttatgtg atctacgtgc aagcagatta cggtgacgat 1080

cccgcagtgg ctctctatac aaagttgggc atacgggaag aagtgatgca ctttgatatc 1140

gacccaagta ccgccaccta acaattcgtt caagccgaga tcggcttccc ggccgcggag 1200

ttgttcggta aattgtcaca acgccgcgaa tatagtcttt actctagata gttaggtact 1260

atagttaatt agctatgatt taagggataa gagatgtaat ttcttatccc tttattatta 1320

gtcatttttc tgctttagcg agtacttttc tactgcagtt aacattcctc ttaataaatt 1380

taattcattg gtttccaatt cggctcgttg atataaccgg cgtagtttca gcataactgc 1440

ttcgttgcga ataaagccaa gttctttata taaacgttcg gtatgggcaa agaaatgttc 1500

taaggcttct gatgtcgggt attctgcaag cggttgaata tcggtttttt cttgcaaatc 1560

tagccaagcc attcggagtt cataacaggc gagttgtacc gccattgcaa gatttaacga 1620

gccatagtcg gggttggttg gaaagtttaa gtgataatgg cattttaata attcttcatt 1680

ggttaagccc acccgttctc gtccaaatac aatcgctact ttaccctgtt cagcaccgct 1740

tgtaagctt 1749

<210> 12

<211> 1194

<212> DNA

<213>Haemophilus parasuis (Haemophilus parasuis)

<400> 12

atgaaaaaaa cactagtagc gttagcagta gcggcatttg cagcatcagc atcagctgta 60

acagtttatg aaaatgaagg tacaaaagtt gattttgatg gtcaattgcg tcttctttta 120

gaaaaacaag cctcaaaagt gaaaggtcaa tcttcaacag atggtggtca cactaactta 180

aagaataata gttctcgttt cggtatttct atcaaacata atatcaatga gaatctctac 240

ggttttggtc gttatgagac tcgccttggc agtggttcta aaaatgctgc aaaatggggt 300

gatgttacaa cagatgaggc ttacgttggt ttaggtggct atggtcatga aatttctttt 360

ggtaaacaag ctgtaatcgg tgatagcatt ggtcaagctg gttttgataa agtatacggt 420

gttggtactg gtggaattaa atatacatat aaggtagata agtctatcac tgtgaacaat 480

caacagggta catttaaata ttcagcacct caagaaggtt ttgatatcct tactcaatct 540

tctgattcag caattaacta tacctataca ggtattgaag gtttgacgtt aggtgctaac 600

tataatgttg caaatgagcg tgagaaggca gatgtaaaag tagattctat taaatctggc 660

tttggtttag gtgctaaata cacagctaag attgcagaaa gtcaatctgt aactgtggca 720

gcaggttata ctcatgatga ctacaaatct ggatctgtta aactaaaagg taaatttgtt 780

gaagcaggtg gtaaatctac agaccatacc tatacagaaa aaccatttaa taagaaagac 840

aaagatggtg tgtactttgg tcttaaatat gtcaacgctc catttactgt agctgttgat 900

ggtggtcatg gtgttgtaaa aacagatgat gttaaagaga aaattaactt cgtaagaact 960

ggcgcaagat ttgatgttac tccaaaatct ggcgtgtatg gaaactactc ttatggtact 1020

tacaaagttg aagatttcaa agcaactgct catcaattca tgttaggtgc agactataaa 1080

ttacataaac aagttgttac ctttgttgaa ggtcgtttaa tcaagaacaa agacagtgat 1140

aacaacaaag ttactgacaa agcacttggt gttggtttac gtgtattatg gtaa 1194

Claims

1. a kind of haemophilus parasuis OmpP2 gene-deleted strains LC Δ OmpP2, belong to 1 type, it is characterised in that deposit number： CCTCC NO：M2017574.

2. haemophilus parasuis OmpP2 gene-deleted strains LC Δs OmpP2 according to claim 1, it is characterised in that opposite In haemophilus parasuis LC strain, the gene of sequence 12 in sequence table has been lacked.

3. haemophilus parasuis OmpP2 gene-deleted strains LC Δs OmpP2 according to claim 1, it is characterised in that opposite In haemophilus parasuis LC strain, virulence reduces.

4. a kind of construction method of haemophilus parasuis OmpP2 gene-deleted strains, it is characterised in that include the following steps：

（1）The upper and lower homology arm of OmpP2 genes is expanded, genetic fragment OmpP2 up and OmpP2 down are obtained；

（2）Amplification gentamicin resistance gene obtains genetic fragment Gm；

（3）Genetic fragment OmpP2 up are connect with Gm to obtain OmpP2 up-Gm, then connect with OmpP2 down to obtain gene piece Section OmpP2 up-down；

（4）Genetic fragment OmpP2 up-down are connected in suicide plasmid pK18mobsacB by digestion, are recombinated Plasmid pK18-OmpP2；

（5）Recombinant plasmid transformed haemophilus parasuis to get.

5. construction method according to claim 4, it is characterised in that step（1）The upper and lower homology arm of middle amplification OmpP2 genes Primer sequence it is as follows：

OmpP2 up-F：5'-ATAGGATCC ACCGCTTGTTCTCTGTATTAAGCGTATAG-3',

OmpP2 up-R：5'-GTTGCTGCTGCGTAACATAATGTTCACCTTAAGTTTTT-3',

OmpP2 down-F：5'-CCAAGTACCGCCACCTAATCTAGATAGTTAGGTACTAT-3',

OmpP2 down-R：5'-TTAAAGCTTACAAGCGGTGCTGAACAGGGTAAAGTAGC-3'.

6. construction method according to claim 4, it is characterised in that step（2）The primer sequence of middle amplification Gm is as follows：

Gm-F:5'-ATGTTACGCAGCAGCAACGATG-3',

Gm-R: 5'-TTAGGTGGCGGTACTTGGGT-3'。

7. the haemophilus parasuis OmpP2 gene-deleted strains LC Δs OmpP2 described in a kind of any one of claim 1-3 or right It is required that the haemophilus parasuis OmpP2 gene-deleted strains that the construction method described in any one of 4-6 is built are in host cell Application in testing and being pathogenic.