CN107586788A - A kind of construction method of pMG36e pgsA gp85 recombinant plasmids - Google Patents

Abstract

The invention provides a kind of construction method of pMG36e pgsA gp85 recombinant plasmids, by the way that gp85 genes and pgsA genes are inserted in Escherichia coli lactic acid bacteria shuttle type expression vector pMG36e, and rotate into the recombinant vector pMG36e pgsA gp85 electricity being prepared in Lactobacillus plantarum engineered strain can successful expression pgsA gp85 fusion proteins, the fusion protein can be widely applied in oral immunity, each phase average daily gain of chicken can be greatly improved, and the ability of chicken body resistance ALV J infection can be improved, so as to be advantageous to increase culture benefit, reduce the risk of ALV J infected chicken bodies.

Description

A kind of construction method of pMG36e-pgsA-gp85 recombinant plasmids

Technical field

The present invention relates to field of biology, specifically provides a kind of structure side of pMG36e-pgsA-gp85 recombinant plasmids Method.

Background technology

J subgroup avian leucosis virus (Avian Leukosis Virus subgroup J, ALV-J) be 1988 first It is isolated from meat type chicken, the main myelocytomatosis and nephroncus for triggering chicken.The disease spreads to rapidly many Country, huge loss is caused to world's aviculture.

It is used to preventing and treating ALV-J infection there is presently no the vaccine of commercialization, it is external mainly by purifying the method for breeder flock Carry out prevention and control ALV-J.But because the overall situation of cultivation has polluted, so rely only on purification is to control ALV-J to infect completely It is impossible.Recent years, many experts and scholars also attempt to develop ALV-J inactivated vaccines and Attenuate vaccine, but due to inactivation disease Also the ability of its inducing antibodies is destroyed while malicious, and the viral antigenic structure is complicated, to expect preferably Attenuate vaccine is very difficult.Therefore, the effective ALV-J vaccines of development of new are imperative.

Because mucous membrane intrusion is the major way of virus and bacterial invasion body, antigen presentation is arrived using probiotics Body mucous membrane tissue, mucosal immune response occurs for induction body, so as to produce specific antibody to resist substantial amounts of bacterium and disease The invasion of poison is highly effective.Lactobacillus plantarum has turned into development focus in recent years as oral vaccine, and it is as mobile load The external source protection antigen gene of body bacterial strain is expressed, by the prebiotic of the immunogenicity of external source target gene and Lactobacillus plantarum Function phase combine, meet new generation vaccine it is cheap, conveniently, it is safe the features such as.And by the immunogenicity of external source target gene and plant The difficult point that the prebiotic function phase of thing lactobacillus combines is from suitable expression vector insertion external source target gene and anchorin Gene, ensure that external source target gene can be effectively in Lactobacillus plantarum surface expression.

PMG36e derives from pWV01 carriers, is to be about by artificial reconstructed pWV01 derivative vectors, plasmid size 3.6kb, it is easy to, to transport entrained by host, foreign protein can be expressed in various bacteria；Succeed at present in breast The expression of other foreign protein genes is carried out in Bacillus, lactococcus and streptococcus.It is by with Lactococcus lactis butterfat It is built-up based on the transcription and translation signal of subspecies protease gene.Plasmid pMG36e is by following main six parts Form：Strong promoter p32, multiple cloning sites, pWV01 replicons, downstream fractional open reading frame, come from lactic acid breast bacterium breast The transcription terminator of the protease gene of fat subspecies and the red toxin resistant gene Emr for coming from plasmid pE194.

1993, Escherichia coli MnSOD was cloned into expression vector pMG36e by Roy etc., and electricity is transformed into carrying respectively In SOD Lactococcus lactis and in shortage SOD Lactobacillus gasseri.1996, Franke etc. was expressed by means of pMG36e LcnD genes, the bacteriocin transhipment merged with beta galactosidase and maturation are closely related, are the transport mechanism of lactococcin Further investigation provides the foundation.2000, Xing etc. also utilized pMG36e clonal expressions mankind's glutathione gene hGSTA1； 2007, Zhang Anyong etc. was successfully built into pMG36e-P30 recombinant vectors using pMG36e, and electricity goes to Lactococcus lactis, was immunized Mouse, as a result confirm that the restructuring oral vaccine can induce mouse specific humoral immunity, promote that cellullar immunologic response occurs.Mesh Before, the expression of a variety of external source target gene is carried out with pMG36e plasmids, enriches relevant lactic acid bacteria genetic engineering and molecule The theoretical foundation of the research of biology, its application are related to multiple key areas (such as food, health care, medicine), also served as simultaneously Important molecular cloning instrument lays the foundation for the further investigation of other related disciplines.

Research finds that ALV-J genomes mainly contain 3 genes：Gag, pol and env.The env gene codes virus of virus Coating, its subgroup specificity is determined by the surface envelope protein of env-gp85 gene codes.Wherein gp85 is responsible for identification Specificity virus acceptor on target cell membrane, belong to the outer membrane protein of virus, be the chondritic of virus surface, it is determined ALSV subgroup specificity, and virus receptor determinant is carried, adhesive attraction is played when infecting permissive cell, and can induce Body produces specific antibody；China is constantly studied ALV-J env gp85 albumen in recent years, 2013, Dou et al. expression ALV-J restructuring gp85 envelope proteins, using CpG adjuvants and Freund's adjuvant and recombinant protein combination vaccinated flock, The maternal antibody detected in chicken body is horizontal, produces preferable immune effect；2014, Zhang et al. reports, by ALV-J weights Group gp85 membrane glycoproteins are immunized after being coated with using liposome, detect chicken body antibody level, are produced preferably immune Effect；Cheng in 2016 etc. is used as vaccine adjuvant and ALV-J restructuring gp85 membrane glycoprotein knots by the use of nano SiO 2 particle Vaccinated flock is closed, achieves certain effect.Above-mentioned related work all provides abundant for follow-up development ALV-J subunit vaccines Theoretical foundation.

Poly-gamma-glutamic acid synzyme A (Poly- γ-Glutamic A, pgsA) comes from bacillus subtilis (Bacillus subtlis), there is a transmembrane region at pgsA N-terminal 26-42 amino acid residues, the transmembrane region exists for It creates condition as bacterium surface displaying element, and foreign protein can be anchored on Host Strains surface by it.The head such as example Nanta Secondary successfully using the enzyme combination different from source of pgsA grappling gene orders, simultaneously successful presentation is to surface of E. coli, because this is Surface display efficiency of uniting is higher, and pgsA grapplings gene is applied to the memebrane protein surface display of gram-positive bacteria by related scholar again Research.Lee etc. is with bacterial strain surface display element pgsA albumen by atypical pneumonia virus (Severe Acute Rwspiratory Syndrome, SARS) associated glycoprotein is expressed in Lactobacillus casei (Lb.casei) surface, and follow-up study shows that the recombinant bacterium can Effectively induce body to produce mucosa-immune, fully evade the drawbacks common of traditional vaccine.Poo etc. is with bacterial strain surface display member The type E7 albumen of Papillomavirus 16 of people is successfully anchored to Lactobacillus casei surface by part pgsA albumen, by the recombinant bacterium mouth The immune C57/BL6 mouse of clothes, successfully induce body to produce mucosa-immune.

In view of the characteristic of pgsA albumen, it has been applied to the surface display of a variety of protokaryon albumen, and particularly it is in lactic acid bacteria Etc. the example for having successful application in Gram-positive F-strain, this studies for us and how is anchored on exogenous medicinal albumen Lactobacillus plantarum cell wall provides theoretical foundation.

And the oral vaccine that the ability for improving the resistance ALV-J infection of chicken body how is obtained using above-mentioned prior art turns into this Field urgent problem to be solved, and recombinant plasmid corresponding to how obtaining is then the most important thing to solve the above problems.

The content of the invention

For blank existing for prior art, the invention provides a kind of structure of pMG36e-pgsA-gp85 recombinant plasmids Method, by the way that gp85 genes and pgsA genes are inserted in Escherichia coli-lactic acid bacteria shuttle type expression vector pMG36e, and will system Standby obtained recombinant vector pMG36e-pgsA-gp85 electricity is rotated into Lactobacillus plantarum engineered strain can successful expression pgsA-gp85 Fusion protein, the fusion protein can be widely applied in oral immunity, can greatly improve each phase average daily gain of chicken, and can carry The ability of high chicken body resistance ALV-J infection, so as to be advantageous to increase culture benefit, reduces the risk of ALV-J infected chicken bodies.

The technical solution adopted in the present invention is：

Two pairs of primers for being directed to gp85 and pgsA genes have been separately designed, have been expanded from J subgroup avian leucosis virus env genes Increase and gp85 envelope glycoprotein genes；Amplified in pgsA from bacillus subtilis full genome, pgsB and pgsC genes PgsA anchorin genes；Wherein its nucleotide sequence of gp85 genetic fragments is as shown in SEQ ID NO.5；PgsA genetic fragments its Nucleotide sequence is as shown in SEQ ID NO.6；

Above-mentioned two GFP is cloned into large intestine-Bacillus acidi lactici shuttle expression carrier pMG36e respectively.Will identification The positive plasmid gp85 and pgsA and expression vector pMG36e gone out is reclaimed with Sac I, Xba I and Hind III single endonuclease digestions respectively Afterwards, connection obtains positive recombinant plasmid pMG36e-pgsA-gp85, and sequencing identification；

Above-mentioned transfer vector plasmid pMG36e-pgsA-gp85 is transferred in Lactobacillus plantarum with electrotransformation, and it is carried out Biological deposits；Using the immunogenicity of Western-blot identification albumen, Flow Cytometry is recycled to detect albumen in bacterium The expression on surface；

By the blue brown Adult cockerel chick in Lactobacillus plantarum oral immunity SPF seas containing recombinant plasmid, detected not after immunity inoculation With period antibody level；Simultaneously after immune 7 weeks, test chicken is carried out to attack malicious protectiveness experiment.

As a result after proving oral immunity chick, the immune response that chicken body produces the anti-ALV-J of specificity can be strengthened；Attack poison Good immune protective effect is shown afterwards.

Further, the concrete technical scheme of the application is as follows：

(1) specific primer is designed：

- the C of PCR primer 5 ' is used in purpose fragment pgsA PCR amplificationsGAGCTCGCGAACTGAGCTTTCATGAAAAG- 3 ', its nucleotide sequence is as shown in SEQ ID NO.1；5′-CTAGTCTAGACTATGATCAATATCAAACGTCA-3 ', its core Nucleotide sequence is as shown in SEQ ID NO.2；

- the TCA of PCR primer 5 ' is used in purpose fragment gp85 PCR amplificationsTCTAGAGGGAGTTCATCTGTTG-3 ', its Nucleotide sequence is as shown in SEQ ID NO.3 and 5 '-TCCAAGCTTATTAGCGCCTGCTAC-3 ', its nucleotide sequence such as SEQ Shown in ID NO.4；

Using above-mentioned primer gp85 envelope glycoprotein genes are amplified from J subgroup avian leucosis virus env genes；From withered PgsA anchorin genes are amplified in pgsA in careless bacillus full genome, pgsB and pgsC genes；

(2) gp85 that step (1) obtains and pgsA genes PCR primer are connected respectively to two single pMD18-T On carrier；

(3) food-grade shuttle expression carrier pMG36e and step (2) carrier T containing gp85 and pgsA genes obtained are used Pass through T4 ligases after restriction enzyme (Sac I, Xba I, Hind III) difference digestion and carry out substep connection；

(4) connection product that step (3) obtains is transformed into Escherichia coli, verified finally by PCR, digestion authentication method Positive colony bacterium；

(5) recombinant vector electricity conversion is entered into Lactobacillus plantarum

(6) expression and immunogenicity of SDS-PAGE and Western-blot technical identification foreign proteins are passed through

(7) detection and localization of the foreign protein on Lactobacillus plantarum surface is verified by Flow Cytometry

The base containing gp85 obtained wherein in step (3) using Xba I with Hind III enzymes to pMG36e carriers and step (2) The carrier T difference digestion of cause, after be attached by T4 ligases, then using Sac I and Xba I enzymes by this connection product The carrier T difference digestion for the gene containing pgsA that PMG36e-gp85 and step (2) obtain, after be attached by T4 ligases, obtain Obtain recombinant plasmid pMG36e-pgsA-gp85；

The recombinant vector pMG36e-pgsA-gp85 electricity that is obtained of the present invention rotates into after Lactobacillus plantarum engineered strain can be into Work(expresses pgsA-gp85 fusion proteins, and the fusion protein can be widely applied in oral immunity, can greatly improve chicken each phase and put down Equal daily gain, and the ability of chicken body resistance ALV-J infection can be improved, so as to be advantageous to increase culture benefit, reduce ALV-J infection The risk of chicken body.

For above-mentioned Lactobacillus plantarum engineered strain, inventor has carried out biological deposits, and preservation information is as follows：

Preservation information

The preservation time：On 06 01st, 2017

Depositary institution's title：China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC

Deposit number：CGMCC No.14209

Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences

Classification And Nomenclature:Lactobacillus plantarum lactobacillus plantarum

Brief description of the drawings

Fig. 1 is restructuring plasmid construction schematic diagram；

Fig. 2 is pcr amplification product electrophoresis schematic diagram,

M is Super DNA Maker in figure；1 and 2 swimming lanes are gp85PCR products in Fig. 2A, 1 and 2 swimming in 936bp, Fig. 2 B Road is pgsA PCR primers, and 1155bp is in the same size with expected results；

The double digestion that Fig. 3 is recombinant plasmid pMG36e-gp85 identifies schematic diagram,

M is DL 5000DNA marker in figure；1 swimming lane is recombinant plasmid pMG36e-gp85 through the and Hind III of Xba I Result after double digestion, 3592bp pMG36e fragments and 930bp gp85 genetic fragments；

The double digestion that Fig. 4 is recombinant plasmid pMG36e-pgsA-gp85 identifies schematic diagram,

M is Super DNA marker in figure；1 swimming lane is recombinant plasmid pMG36e-pgsA-gp85 through the and Xba of Sac I The pMG36e-gp85 fragments of result after I double digestion, about 4500bp and 1143bp pgsA genetic fragments；

Fig. 5 is recombinant plasmid pMG36e-pgsA-gp85 PCR the results,

M is 1kb Ladder in Fig. 5 A；1 swimming lane is gp85PCR products；M is Super DNA marker in Fig. 5 B；1 swimming Road is pgsA PCR primers；

Fig. 6 is recombination fusion protein pgsA-gp85 (not purifying) SDS-PAGE result schematic diagrams,

M is pre- dsred protein Marker in figure；1 is recombination fusion protein pgsA-gp85, and it is 69KD；

Fig. 7 is the Western blot results of recombinant protein,

M is pre- dsred protein Marker in figure；1 is recombination fusion protein pgsA-gp85, and it is 69KD；

Fig. 8 is flow cytomery histogram,

Wherein detect A, B, D, mouse IgG as secondary antibody by the use of monoclonal antibody JE9 as primary antibody, the mouse IgG of FITC marks respectively C is detected as secondary antibody, A is Lactobacillus plantarum in figure；B is pMG36e-gp85 recombinant plant lactobacillus；C and D is pMG36e- PgsA-gp85 recombinant plant lactobacillus, the P1 in figure partly represent the flora chosen, and P3 parts represent that the fluorescence of surface protein is strong Degree；

Fig. 9 is the amount of fluorescence column schematic diagram of P3 part surface albumen in Fig. 8；

Figure 10 is specific IgG level in oral immunity chicken serum；

Figure 11 is that sIgA is horizontal in oral immunity chicken bile, duodenum washing lotion and serum；A：The horizontal B of sIgA in bile: The horizontal C of sIgA in duodenum washing lotion:SIgA is horizontal in serum；

Figure 12 is immune rear each group chicken body weight testing result.

Embodiment

In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and It is not used in the restriction present invention.

Escherichia coli (E.coli) DH5 α competent cells are purchased from TaKaRa biotech companies, Lactobacillus plantarum (Lactobacillus plantarum) HQ542228 bacterial strains are purchased from Chinese industrial Microbiological Culture Collection administrative center； pMD18-T Vector Cloning Kit；PMG36e vector kits are purchased from TaKaRa biotech companies；In restricted Enzyme cutting Sac I, Xba I and Hind III, T4 DNA Ligase and Taq archaeal dna polymerase all have purchased from Takara biotechnologys Limit company.

The amplification of the gp85 genes of embodiment 1 and pgsA genes

(1) specific primer is designed：

- the C of PCR primer 5 ' is used in purpose fragment pgsA PCR amplificationsGAGCTCGCGAACTGAGCTTTCATGAAAAG- 3 ', its nucleotide sequence is as shown in SEQ ID NO.1；5′-CTAGTCTAGACTATGATCAATATCAAACGTCA-3 ', its core Nucleotide sequence is as shown in SEQ ID NO.2；

- the TCA of PCR primer 5 ' is used in purpose fragment gp85 PCR amplificationsTCTAGAGGGAGTTCATCTGTTG-3 ', its Nucleotide sequence is as shown in SEQ ID NO.3 and 5 '-TCCAAGCTTATTAGCGCCTGCTAC-3 ', its nucleotide sequence such as SEQ Shown in ID NO.4；

Using above-mentioned primer gp85 envelope glycoprotein genes are amplified from J subgroup avian leucosis virus env genes；From withered PgsA anchorin genes are amplified in pgsA in careless bacillus full genome, pgsB and pgsC genes；As a result such as Fig. 2 It is shown；

Gp85 PCR amplification system and condition：With pMD18T-env (Limei Zhang, Dongjie Cai, Xiaona Zhao,et al.Lipsomes containing recombinant gp85protein vaccine against ALV-J in chickens[J].Vaccine,2014,32:It is 2452-2456.) template, entering performing PCR with gp85 primers expands：

Using 20 μ l reaction system:10×PCR Buffer 2.4μl；dNTP 2μl(2.5mM)；Upstream and downstream primer is each lμl(25umol/L)；The μ l of recombinant plasmid template 1；The μ l of Taq archaeal dna polymerases 0.6 (1U/ μ l)；Deionized water is supplemented to 11.4 μ l. PCR loop parameter is:94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s；58 DEG C of 60s annealing；72 DEG C of extension l min, totally 30 are followed Ring, last 72 DEG C of extensions 10min.

The gp85 fragments obtained, its nucleotide sequence is as shown in SEQ ID NO.5；

PgsA PCR amplification system and condition：With pgsA, pgsB and pgsC (Chun-Hua Wei, Jian-Kui Liu, Xi-Lin Hou,et al.Immunogenicity and protective efficacy of orally or intranasally administered recombinant Lactobacillus casei expressing ETEC K99 [J].Vaccine.2010,28:It is 4113-4118.) template, entering performing PCR with pgsA primers expands：

Using 20 μ l reaction system：The μ l of plasmid template 1；Each l μ l (25umol/L) of upstream and downstream primer；Taq DNA polymerize The μ l of enzyme 0.6 (1U/ μ l)；dNTP 2μl(2.5mM)；10×PCR Buffer 2.4μl；Deionized water is supplemented to 11.4 μ l.PCR Loop parameter be arranged to：94 DEG C of 5min pre-degenerations；94 DEG C of 30s denaturation；58 DEG C of 60s annealing；72 DEG C of l min 12s extensions, altogether 30 circulations, last 72 DEG C of extensions 10min.

The pgsA fragments obtained, its nucleotide sequence is as shown in SEQ ID NO.6；

Embodiment 2

The gp85 that step (1) obtains and pgsA genes PCR primer are connected respectively to two single pMD18-T carriers On；

PMD18-T carriers and two target gene fragments of purifying recovery are attached respectively, linked system is PMD18-T carriers 0.5 μ l, Solution I 5.0 μ l, the μ l of purified pcr product 4.5.Connection product is mixed, slightly centrifuges removal Bubble, 16 DEG C of connection 8-10h are positioned over, 4 DEG C is then put in and saves backup.

The recombinant plasmid pMG36e-pgsA-gp85 of embodiment 3 acquisition

Expression vector pMG36e and the double digestion of gp85 target gene fragments reclaim

Expression vector pMG36e and target gene gp85 fragments are respectively through Hind III and the double digestions of Xba I.Using 50 μ l's Digestion system：10 × M Buffer, 6 μ l；Each 3 μ l of Xba I and Hind III；gp85/pMG36e 15μl；Supplement ddH₂O 23μl。 Reactant is mixed according to above system, 37 DEG C of water-bath 3h.PMG36e carrier segments and mesh are reclaimed with glue reclaim kits Fragment gp85.After product recovery purifying recovering state is observed with 1% agarose gel electrophoresis.

Target gene gp85 and expression vector pMG36e connection converts

A. connect：The digestion carrier segments of glue reclaim are attached with digestion purpose fragment.Using 20 μ l linked systems： pMG36e 8μl；The μ l of gp85 fragments 8；T4 DNA Ligase 2μl；10×T4 ligase Buffer 2μl.By above material After mixing, connected overnight in 16 DEG C of connection instrument.

B. convert：The DH5 α competent cells frozen are taken out from -70 DEG C of ultra low temperature freezers, it is fast in mixture of ice and water It is quick-frozen to melt, aseptically, 10 μ l connection products and competent cell are mixed, ice bath 30min is then placed in 42 DEG C of constant temperature Water-bath heat shock 90s, after take out ice bath 2min immediately, 1mL LB fluid nutrient mediums are rapidly added afterwards, on 37 DEG C of shaking tables 150r concussions incubate culture 45min, and culture 5000r/min is centrifuged into 5min after taking-up, supernatant discarding, leaves behind about 100 μ l Bacterium solution, piping and druming are applied to LB flat boards (g/mL of μ containing Emr+300), are inverted and are incubated overnight in 37 DEG C of incubators of placement after mixing.

Recombinant plasmid pMG36e-gp85 extraction

Use the small extraction reagent kit extraction plasmid of TIANGEN DP103 plasmids

From reformer plate, the good single bacterium colony of picking growth conditions, aseptic inoculation to 5mL Emr+LB containing 300ug/mL liquid In body culture medium, after 37 DEG C of shaken cultivation 12h, bacterium solution extraction plasmid is collected, operating procedure is specific as follows：

A. column equilibration step：Into adsorption column CP3, (adsorption column is put into collecting pipe) adds 500 μ l equilibrium liquid BL, 12000rpm centrifuges 1min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.

B. the bacterium solution for having cultivated 12-16h is collected, draws 1.5mL bacterium solutions in sterile l.5mL centrifuge tube, 12000r/min 1min is centrifuged, supernatant discarding, is so repeated 2-3 times, collects thalline；

C. 250 μ l solution P1 (please first check whether and added RNaseA) are added into centrifuge tube, use vortex oscillator Or the thorough suspended bacterial precipitation of pipettor；

D. 250 μ l solution P2 are added into centrifuge tube, leniently spinning upside down 6-8 times makes thalline fully crack.

E. 350 μ l solution P3 are added into centrifuge tube, are leniently spun upside down 6-8 times immediately, fully mixes, now occurs White flock precipitate, 12000r/min centrifugations 10min；

The supernatant that previous step is collected is transferred to adsorption column CP3 (adsorption column is put into collecting pipe) by f with pipettor, is paid attention to Try not to suction out precipitation, 12000r/min centrifugation 1min, outwell the waste liquid in collecting pipe, adsorption column CP3 is put into collecting pipe In；

G. 500 μ l protein liquid removals PD, 12000r/min centrifugation 1min are added into adsorption column CP3, are outwelled in collecting pipe Waste liquid, adsorption column CP3 is placed back in collecting pipe；

H. 600 μ l rinsing liquids PW (please first check whether and added absolute ethyl alcohol), 12000r/ are added into adsorption column CP3 Min centrifuges 1min, outwells the waste liquid in collecting pipe, adsorption column CP3 is put into collecting pipe；

I. repeat step 8；

J. adsorption column CP3 is put into collecting pipe, 12000r/min centrifugations 2min, it is therefore an objective to float the residual in adsorption column Washing lotion removes；

K. adsorption column CP3 is placed in a clean centrifuge tube, 50-100 μ l elutions is added dropwise to adsorbed film middle part Buffer solution EB, room temperature place 2min, and plasmid solution is collected into centrifuge tube by 12000r/min centrifugations 2min.

1% agarose nucleic acid gel electrophoresis, after observation identification, it can be directly used for subsequent experimental or be placed in -20 DEG C of refrigerators Preserve.

Recombinant plasmid pMG36e-gp85 identification

A. the pMG36e-gp85PCR identifications of recombinant plasmid

Under sterile working, picking single bacterium colony, it is inoculated in 10mL LB fluid nutrient mediums (Emr+ containing 300ug/mL), 37 DEG C Extract plasmid after culture 14h, using 1% agarose nucleic acid gel electroresis appraisal for positive pMG36e-gp85 recombinant plasmids as Template, enter performing PCR identification.From 20 μ l PCR reaction systems：The μ l of recombinant plasmid dna 0.5；Each 1 μ l of gp85 upstream and downstream primers； dNTP 2μl；The μ l of Taq archaeal dna polymerases 0.6；10×PCR Buffer 2.4μl；Supplement ddH₂O 11.9μl。

The PCR reaction cycle parameters set are as follows：94 DEG C of 5min of pre-degeneration；It is denatured 94 DEG C of 30s；Anneal 58 DEG C of 60s；Prolong 72 DEG C of l min are stretched, totally 30 circulations, finally extend 72 DEG C of 10min.The identification PCR productions of 1% agarose nucleic acid gel electrophoresis observation Thing, as shown in Fig. 5 A swimming lanes 1：About 936bp gp85 PCR primers.

B. recombinant plasmid pMG36e-gp85 double digestion identification

Recombinant plasmid pMG36e-gp85 after PCR is identified carries out double digestion through Hind III and Xba I, and identification uses 10 μ l reaction systems：The μ l of recombinant plasmid dna 6；Each 0.25 μ l of Hind III and Xba I；10×M Buffer 1μl；ddH2O 2.5μ l.Reactant is mixed by above-mentioned system, after 37 DEG C of effect 3h, 1% agarose nucleic acid gel electrophoresis observation identification, as a result such as Fig. 3 It is shown:3592bp pMG36e fragments and 936bp gp85 genetic fragments.

Expression vector pMG36e-gp85 and the double digestion of target gene pgsA fragments reclaim

Expression vector pMG36e-gp85 and target gene pgsA fragments are respectively through Sac I and the double digestions of Xba I.Using 60 μ L digestion system：Each 15 μ l of pMG36e-gp85/pgsA；Each 3 μ l of Sac I and Xba I；10 × M Buffer, 6 μ l；Supplement ddH₂O 33μl.Reactant is mixed according to above system, 37 DEG C of water-bath 3h.Glue reclaim kit recovery purifying pMG36e- Gp85 carrier segments and purpose fragment pgsA.With the recovering state of 1% agarose nucleic acid gel electrophoresis observation product after purification.

Target gene pgsA and expression vector pMG36e-gp85 connection converts

A. connect：The carrier segments reclaimed after digestion and target gene fragment are attached.Using 10 μ l linked systems： pMG36e-gp85 4μl；The μ l of pgsA fragments 4；T4 DNA Ligase 1μl；10×T4 ligase Buffer 1μl.By more than After material mixes according to the above ratio, 16 DEG C of connection instrument connections are overnight.

B. convert：The DH5 α competence frozen is taken out from -70 DEG C of ultra low temperature freezers, it is rapid to freeze in mixture of ice and water Melt, aseptically, 10 μ l connection products and competent cell are mixed, ice bath 30min is then placed in 42 DEG C of waters bath with thermostatic control Pot heat shock 90s, after take out ice bath 2min immediately, be rapidly added 1mL LB fluid nutrient mediums afterwards, the 150r on 37 DEG C of shaking tables Concussion incubates culture 45min, and culture 5000r/min is centrifuged into 5min after taking-up, supernatant discarding, leaves behind about 100 μ l bacterium solutions, Piping and druming is applied to LB flat boards (containing Emr+300ug/ml) after mixing, be inverted and be incubated overnight in 37 DEG C of incubators of placement.

Recombinant plasmid pMG36e-pgsA-gp85 extraction

Use the small extraction reagent kit extraction plasmid of TIANGEN DP103 plasmids

From reformer plate, the good single bacterium colony of picking growth conditions, aseptic inoculation to 5mL Emr+LB containing 300ug/mL liquid In body culture medium, after 37 DEG C of shaken cultivation 12h, bacterium solution extraction plasmid is collected, operating procedure is specific as follows：

A. column equilibration step：Into adsorption column CP3, (adsorption column is put into collecting pipe) adds 500 μ l equilibrium liquid BL, 12000rpm centrifuges 1min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.

B. the bacterium solution for having cultivated 12-16h is collected, draws 1.5mL bacterium solutions in sterile l.5mL centrifuge tube, 12000r/min 1min is centrifuged, supernatant discarding, is so repeated 2-3 times, collects thalline；

C. 250 μ l solution P1 (please first check whether and added RNaseA) are added into centrifuge tube, use vortex oscillator Or the thorough suspended bacterial precipitation of pipettor；

D. 250 μ l solution P2 are added into centrifuge tube, leniently spinning upside down 6-8 times makes thalline fully crack.

E. 350 μ l solution P3 are added into centrifuge tube, are leniently spun upside down 6-8 times immediately, fully mixes, now occurs White flock precipitate, 12000r/min centrifugations 10min；

F. the supernatant that previous step is collected is transferred to adsorption column CP3 (adsorption column is put into collecting pipe) with pipettor, noted Meaning tries not to suction out precipitation, 12000r/min centrifugation 1min, outwells the waste liquid in collecting pipe, adsorption column CP3 is put into collection Guan Zhong；

G. 500 μ l protein liquid removals PD, 12000r/min centrifugation 1min are added into adsorption column CP3, are outwelled in collecting pipe Waste liquid, adsorption column CP3 is placed back in collecting pipe；

H. 600 μ l rinsing liquids PW (please first check whether and added absolute ethyl alcohol), 12000r/ are added into adsorption column CP3 Min centrifuges 1min, outwells the waste liquid in collecting pipe, adsorption column CP3 is put into collecting pipe；

I. repeat step 8；

J. adsorption column CP3 is put into collecting pipe, 12000r/min centrifugations 2min, it is therefore an objective to float the residual in adsorption column Washing lotion removes；

K. adsorption column CP3 is placed in a clean centrifuge tube, 50-100 μ l elutions is added dropwise to adsorbed film middle part Buffer solution EB, room temperature place 2min, and plasmid solution is collected into centrifuge tube by 12000r/min centrifugations 2min.

1% agarose nucleic acid gel electrophoresis, after observation identification, it can be directly used for subsequent experimental or be placed in -20 DEG C of refrigerators Preserve.

Recombinant plasmid pMG36e-pgsA-gp85 identification

A. the pMG36e-pgsA-gp85PCR identifications of recombinant plasmid

Under sterile working, picking single bacterium colony, it is inoculated in 10mL LB fluid nutrient mediums (Emr+ containing 300ug/mL), 37 DEG C Plasmid is extracted after culture 14h, the pMG36e-pgsA-gp85 recombinant plasmids that gel electrophoresis is accredited as the positive are carried out as template PCR is identified.Using 20 μ l reaction system：The μ l of recombinant plasmid dna 0.5；Each 1 μ l of pgsA upstream and downstream trip primer；Taq DNA gather The μ l of synthase 0.6；dNTP 2μl；10×PCR Buffer 2.4μl；Supplement the μ l of ddH2O 11.9.

The PCR reaction cycle parameters set are as follows：94 DEG C of 5min of pre-degeneration；It is denatured 94 DEG C of 30s；Anneal 58 DEG C of 60s；Prolong 72 DEG C of l min 12s are stretched, totally 30 circulations, finally extend 72 DEG C of 10min.Identification PCR productions are observed with 1% agarose gel electrophoresis Thing, as shown in Fig. 5 B swimming lanes 1：About 1155bp pgsA PCR primers.

B. recombinant plasmid pMG36e-pgsA-gp85 digestion identification

Recombinant plasmid after PCR is identified carries out double digestion through Sac I and Xba I, and identification uses 10 μ l reaction systems：Weight Group DNA 6 each 0.25 μ l of μ l Sac I and Xba I；10×M Buffer 1μl；ddH2O 2.5μl.Reactant press with Upper system mixes, after 37 DEG C of effect 3h, the identification of 1% agarose nucleic acid gel electrophoresis observation, as a result as shown in Figure 4：About 4500bp PMG36e-gp85 fragments and 1143bp pgsA genetic fragments.

The structure of the pMG36e-pgsA-gp85 recombinant plant lactobacillus of embodiment 4

The preparation of Lactobacillus plantarum competent cell

A. Lactobacillus plantarum HQ542228 is taken out from -70 DEG C of refrigerators, is connect in being rule on the MRS solid mediums of non-resistant Kind, 37 DEG C of Anaerobic culturel 12h；

B. on the MRS flat boards after line overnight, picking single bacterium colony, be inoculated in 5mL MRS fluid nutrient mediums 37 DEG C it is quiet Only overnight incubation；

C. culture is obtained into product next day in 2% ratio, be inoculated in 50mL MRS fluid nutrient mediums, 37 DEG C of static trainings Support；

D. when culture OD600 is up to 0.5~0.6, culture is put into 10~15min on ice, dispenses 2mL centrifuge tubes, 4000r/min centrifugations 10min collects thalline；

E. washing lotion (0.15mmol/L magnesium chlorides and 5mmol/L) sodium phosphate of precooling is used) lightly precipitation is suspended, 4000r/min centrifuges 10min, such repeated washing 3 times；

F. with the suspensions (0.15mmol/L magnesium chlorides, 5mmol/L sodium phosphates and 0.3mmol/L sucrose) of 50 μ l precoolings gently Precipitation is suspended, -70 DEG C save backup or are directly used in electricity and turn.

Recombinant plasmid electricity is transformed into Lactobacillus plantarum

A. take the μ l of recombinant plasmid pMG36e-pgsA-gp85 3 to add in Lactobacillus plantarum competent cell, stand on ice 15min；

B., the Lactobacillus plantarum competent cell for being mixed with plasmid is all added to dry 2mm electric shock cups (to be immersed in advance 75% ethanol) in, 10min is stood on ice；

C. electric shock cup is dried with filter paper, be put into electric conversion instrument, by 2.0KV voltages, 25uF electric capacity, 400 Ω resistance Etc. parameter Rapid pulses of electricity, burst length 4ms；

D. after electric pulse, added into electric shock cup contain 0.5% glycine MRS fluid nutrient medium 1mL immediately, after mixing, entirely Portion is transferred in Eppendorf pipes, 37 DEG C of static gas wave refrigerator 1h；

E. 50 μ l cultures are taken to be spread evenly across in the MRS solid mediums of erythromycin containing 5ug/mL, 37 DEG C of static anaerobism trainings 12-24h is supported i.e. it can be seen that single bacterium colony.

The protein expression and Western-blot of the pMG36e-pgsA-gp85 recombinant plant lactobacillus plasmids of embodiment 5

As a result

The pMG36e-pgsA-gp85 recombinant plants lactobacillus identified is inoculated in 1mL MRS fluid nutrient mediums by 2% In, 37 DEG C of static gas wave refrigerators are stayed overnight.The bacterium solution of activation is inoculated in 10mL MRS culture mediums by next day by 2%.37 DEG C of static gas wave refrigerators 6h or so, start to sample when bacterium solution OD600 is about 1.0.2mL samples are taken, 7000r/min centrifugations 1min collects thalline, adds l 0mg/mL lysozymes 400 μ l, 37 DEG C of water-bath 30min, 7000r/min centrifugation 1min collect thalline, 4 × sample-loading buffer of addition (containing DTT), mixed with whirlpool oscillator, 10min is boiled in boiling water bath, l 0000r/min centrifugation 5min, takes 15 μ l supernatants, with 10% separation gel 10mL, 5%, which concentrates glue 3mL, carries out carrying out SDS polyacrylamine gels electrophoresis (SDS-PAGE) with glue.

By without the broken direct loading of expressing protein thalline, after carrying out SDS-PAGE, according to half dry type electrophoretic blotting groove Operation instruction, carry out transferring film.Using mouse resource monoclonal antibody (MAb JE9) as first antibody, the sheep anti-mouse igg of HRP marks (1：6000) secondary antibody is used as, carries out conventional Westen-blot detections.Operating procedure is specific as follows：

A. cut the thick filter paper of 6 pieces of 3mm and 1 piece of pvdf membrane, its size are consistent with gel to be turned.Pvdf membrane is soaked with methanol, Together it is put into again with the filter paper sheared in transfering buffering liquid after activation processing 15s and soaks 30min；

B. it is sequentially placed on the plate of transfer groove：One layer of sponge, two layers of filter paper, protein adhesive, pvdf membrane, two layers of filter paper, often One layer will drain bubble (using glass bar), and it is noted that pvdf membrane and protein adhesive, which are greater than filter paper, prevents short circuit, cover negative electrode Cover, 200v, 110mA, transferring film 90min, the protein in gel is transferred on pvdf membrane；

C. after the completion of transferring film, the film shifted is put into TBST cleaning solutions, rinses 3 times, 1 7min, is immersed after taking-up In 5% skimmed milk power, in slowly being shaken on horizontal shaker, 4 DEG C are closed 90min or stayed overnight；

D. next day, film is washed with TBST solution, washes 3 times, 1 7min, film is immersed into l respectively:500 dilution mouse source positive serums In, room temperature slowly shakes 2h；

E. wash film with TBST solution, wash 3 times, 1 7min, after film immersed into l respectively：The goat-anti of 2000 dilution HRP marks In mouse IgG positive serums, room temperature slowly shakes 2h；

F. film is washed with TBST solution, washes 3 times, 1 7min, finally film is operated and observed with DAB colour reagent boxes And film recording experimental result as a result,.

Recombinant expression carrier pMG36e-pgsA-gp85 is converted into Lactobacillus plantarum, by thalline after quiescent culture expression Direct loading carries out SDS-PAGE analyses, it was demonstrated that thalline is correctly expressed, and expressing protein is about 69KD sizes, as a result sees Fig. 6 Swimming lane 1.

Pvdf membrane after the completion of transferring film, the sheep marked respectively with primary antibody mouse resource monoclonal antibody (MAb JE9), secondary antibody HRP Anti- mouse IgG (1:6000) after acting on, develop the color, can be observed by the operating instruction in DAB colour reagent boxes, recombinant bacterium is in expected position The band for about 69KD occurred is put, it is and estimated in the same size, as a result see Fig. 7 swimming lanes 1.

The flow cytometry of embodiment 6 is to detection and localization result of the foreign protein on Lactobacillus plantarum surface

Detect Lactobacillus plantarum, pMG36e-gp85/Lb.plantarum recombinant plant breast bars respectively using flow cytometry Bacterium and the fluorescence intensity and quantity of pMG36e-pgsA-gp85/Lb.plantarum recombinant plant lactobacillus bacterial surface proteins.

By pMG36e-pgsA-gp85 recombinant plants lactobacillus, pMG36e-gp85 recombinant plants lactobacillus and plant breast bar Bacterium cultivates 6h respectively, and whether the foreign protein for identifying expression using flow cytometry is positioned at Lactobacillus plantarum cell surface, with It is specific operating procedure down：

A. Lactobacillus plantarum, pMG36e-gp85 recombinant plants lactobacillus and pMG36e-pgsA-gp85 recombinant plants breast are taken Each l mL of bacillus bacterium solution, are collected by centrifugation thalline, and PBS is washed three times, with 200 mesh sieve net filtrations；

B. it is 3 × 10 with PBS adjustment cell concentrations⁴Supernatant is abandoned in individual/mL, centrifugation, adds 5% skimmed milk, 4 DEG C of closings overnight；

C. supernatant discarding is centrifuged, PBS is washed three times, adds monoclonal antibody JE9,4 DEG C of effect 5h；

D.PBS washs the IgG fluorescence secondary antibodies (l for three times, adding FITC mark sheep anti mouses:100 dilutions), 4 DEG C of lucifuge effects 5h；

Loading after e.PBS washings three times, flow cytomery (FITC atmosphere ion excitation fluorescence, launch optical wavelength For 525nm, excitation light wave wavelength is 488nm, produces green fluorescence), preserve picture, analysis result.

As a result as shown in the P3 parts in Fig. 8 and Fig. 9, pMG36e-pgsA-gp85/Lb.plantarum recombinant plants breast The fluorescence intensity and quantity of bacillus (D) surface protein are obviously higher than Lactobacillus plantarum (A), pMG36e-gp85/ Lb.plantarum recombinant plants lactobacillus (B) and the pMG36e-pgsA- for not adding the anti-igg of mouse source two of FITC marks to treat Gp85/Lb.plantarum recombinant plants lactobacillus (C), and A, B, C three compare, the fluorescence intensity and quantity of surface protein are equal No significant difference.The above results illustrate that pgsA anchorins gene is successfully expressed ALV-J gp85 antigen proteins in bacterium table On face.

The potency of ALV-J gp85 specific IgGs in the serum of embodiment 7

As shown in table 1, using the positive restructuring pMG36e-pgsA-gp85 Lactobacillus plantarums identified as experimental group, using plant Thing lactobacillus is negative control group, and PBS is that blank control group progress oral immunity is commented its anti-ALV-J immune effect Valency, the serum antibody IgG potency of recombinant plant lactobacilli vaccines immunized chickses is determined, in bile, duodenum washing lotion and serum SIgA potency, the change of body weight and the positive detection rate for attacking malicious restrovirus mass formed by blood stasis carry out the oral recombinant plant lactobacillus epidemic disease of comparison Immune protective effect of the seedling to ALV-J.

The animal packet of table 1 and immunizing dose

As a result it is as follows：

7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d, 70d, 77d, 84d after initial immunity, each group difference wing are quiet Arteries and veins is taken a blood sample, and detects specific ALV-J gp85IgG antibody titers in serum.Test result indicates that (Figure 10)：Experimental group ALV-J Gp85IgG antibody levels are apparently higher than control group, and conspicuousness occur in experimental group ALV-J gp85IgG antibody levels after head exempts from Raise (p ＜ 0.05), and after the third immunization, ALV-J gp85IgG antibody titers reach highest.And control group antibody water Flat change is little；

7d, 14d, 21d, 28d, 35d, 42d, 49d after initial immunity, each group take respectively bile, duodenum washing lotion and Serum carries out the qualitative detection of ALV-J Specific IgA antibodies.Result of the test shows：In each stage detection, experimental group bile, ten The testing result of the ALV-J Specific IgA antibodies of two duodenum 12 washing lotions and blood serum sample is positive, and control group bile, 12 The testing result of the ALV-J Specific IgA antibodies of duodenum 12 washing lotion and blood serum sample is negative.It is specific as shown in table 2：

The ALV-IgA antibody positive rates of each group chicken bile of table 2, duodenum washing lotion and blood serum sample

Chicken bile after the qualitative detection for carrying out ALV-J specificity IgA, duodenum washing lotion and blood serum sample are carried out The quantitative detection of sIgA antibody.Result of the test shows (Figure 11)：SIgA in experimental group bile, duodenum washing lotion and blood serum sample Antibody titer is above control group, and experimental group sIgA antibody levels after head exempts from are significantly raised (p ＜ 0.05), and the 3rd Secondary immune rear antibody titer peaks.Control group OD value changes are little；

Each group was weighed in weekly after initial immunity, until end of weighing in the 12nd week.From every group of body weight result It can be seen that (Figure 12)：Oral recombinant plant lactobacillus group, orally taken plant lactobacillus group weight levels are always above blank control Group, and there is conspicuousness growth in 35d, 42d and 56d relative to blank control group, the body weight of orally taken plant lactobacillus group, And there is conspicuousness growth in 35d, 42d, 56d, 70d, 77d and 84d in the body weight of oral recombinant plant lactobacillus group.Control The weight levels of group are always below oral recombinant plant lactobacillus group and orally taken plant lactobacillus group；

Orally taken plant lactobacillus group, oral recombinant plant lactobacillus group and attack malicious control group attacked after end is exempted from poison and even Carry out viremia virusemia detection within continuous 4 weeks, then count positive rate；The 3rd week control group has chicken dead after poison is attacked, according to cut open inspection Change, is diagnosed as avian leukosis.The protective rate of oral recombinant plant lactobacillus group is all higher than orally taken plant lactobacillus group and attacks poison Control group, and there is significant difference (p ＜ 0.05), it is specific as shown in table 3：

Table 3 attacks each group viremia virusemia positive rate after poison

Result above shows that recombinant plant lactobacillus can significantly improve serum moderate resistance ALV-J's as vaccine immunity chick The IgA antibody potency of IgG antibody potency and bile, duodenum washing lotion and serum moderate resistance ALV-J, promote body weight increase and attack poison Good immune protective effect is shown afterwards.

Sequence table

<110>Shandong Agricultural University

<120>A kind of construction method of pMG36e-pgsA-gp85 recombinant plasmids

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 30

<212> DNA

<213>Artificial sequence ()

<400> 1

cgagctcgcg aactgagctt tcatgaaaag 30

<210> 2

<211> 32

<212> DNA

<213>Artificial sequence ()

<400> 2

ctagtctaga ctatgatcaa tatcaaacgt ca 32

<210> 3

<211> 25

<212> DNA

<213>Artificial sequence ()

<400> 3

tcatctagag ggagttcatc tgttg 25

<210> 4

<211> 24

<212> DNA

<213>Artificial sequence ()

<400> 4

tccaagctta ttagcgcctg ctac 24

<210> 5

<211> 936

<212> DNA

<213>Artificial sequence ()

<400> 5

tctagaggag ttcatctgtt gcaacaacca ggaaacgtgt gggtcacctg ggcgaatatg 60

acgggccgaa cagatttttg ccttagtcta cagtcagcga cctcaccatt ccgcacctgc 120

ttgataggca ttccacagta tcctctgaac acctttgagg gatatgtcac taatgttact 180

gcttgcgaaa acaacgccga tttagccaac caaacagcat gcttgataaa ggctctaaac 240

acaaccctcc cttgggaccc ccaagaattg gatattttag ggtctcagat gatcaagaac 300

ggaacaacac gtacgtgtgt tacctttggt tcgatgtgct ataaagagaa caatcacagc 360

agagtctgcc acaattttga tgggaatttt aatgggactg gtggggtgga agcagaattg 420

cgtgacctca tagcaaaatg gggcaaaggt gaccctcgta taagacccta tgtcaaccaa 480

tcatggacga tggtaagtcc aataaacaca gagagttttt caataagtag tagatattgt 540

ggattcacca gtaacgagac tcgttactat gaagggaact tttctaattg gtgcagttca 600

aaaggggggg aatggtcagt ggggtacagc aacgggacag attgttccag caacacgacg 660

gattgcggtg gtaattgtac agcggaatgg aattattatg catatgggtt taccttcggg 720

ggtaagccag agatattgtg gaataatggg actgctaagg cactcccacc aggtattttc 780

ttgatttgtg gggacagggc ttggcaaggc atcccgcgta acgccttggg agggccctgt 840

tatctaggac aattgactat gctctctcct aactttacca cctggataac atatgggccg 900

aacattacgg gtcaccgccg tagcaggcgc aagctt 936

<210> 6

<211> 1155

<212> DNA

<213>Artificial sequence ()

<400> 6

gagctcatga aaaaagaact gagctttcat gaaaagctgc taaagctgac aaaacagcaa 60

aaaaagaaaa ccaataagca cgtatttatt gccattccga tcgtttttgt ccttatgttc 120

gctttcatgt gggcgggaaa agcggaaacg ccgaaggtca aaacgtattc tgacgacgta 180

ctctcagcct catttgtagg cgatattatg atgggacgct atgttgaaaa agtaacggag 240

caaaaagggg cagacagtat ttttcaatat gttgaaccga tctttagagc ctcggattat 300

gtagcaggaa actttgaaaa cccggtaacc tatcaaaaga attataaaca agcagataaa 360

gagattcatc tgcagacgaa taaggaatca gtgaaagtct tgaaggatat gaatttcacg 420

gttctcaaca gcgccaacaa ccacgcaatg gattacggcg ttcagggcat gaaagatacg 480

cttggagaat ttgcgaagca aaatcttgat atcgttggag cgggatacag cttaagtgat 540

gcgaaaaaga aaatttcgta ccagaaagtc aacggggtaa cgattgcgac gcttggcttt 600

accgatgtgt ccgggaaagg tttcgcggct aaaaagaata cgccgggcgt gctgcccgca 660

gatcctgaaa tcttcatccc tatgatttca gaagcgaaaa aacatgctga cattgttgtt 720

gtgcagtcac actggggcca agagtatgac aatgatccaa acgaccgcca gcgccagctt 780

gcaagagcca tgtctgatgc gggagctgac atcatcgtcg gccatcatcc gcacgtctta 840

gaaccgattg aagtatataa cggaaccgtc attttctaca gcctcggcaa ctttgtcttt 900

gaccaaggct ggacgagaac aagagacagt gcactggttc agtatcacct gaagaaaaat 960

ggaacaggcc gctttgaagt gacaccgatc gatatccatg aagcgacacc tgcacctgtg 1020

aaaaaagaca gccttaaaca gaaaaccatt attcgcgaac tgacgaaaga ctctaatttc 1080

gcttggaaag tagaagacgg aaaactgacg tttgatattg atcatagtga caaactaaaa 1140

tctaaataat ctaga 1155

Claims (2)

1. A kind of 1. pMG36e-pgsA-gp85 recombinant plasmids, it is characterised in that：It contains gp85 and pgsA genetic fragments, wherein Its nucleotide sequence of gp85 genetic fragments is as shown in SEQ ID NO.5；Its nucleotide sequence of pgsA genetic fragments such as SEQ ID Shown in NO.6.
2. 2. the answering in the Lactobacillus plantarum containing recombinant plasmid is prepared of pMG36e-pgsA-gp85 recombinant plasmids described in claim 1 With.