CN110358782A

CN110358782A - A kind of polygenes recombination Lactobacillus casei, preparation method and application

Info

Publication number: CN110358782A
Application number: CN201910668557.5A
Authority: CN
Inventors: 杨霞; 常洪涛; 王新卫; 刘红英; 李永涛; 赵军; 陈陆; 王坤芃; 王川庆
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2019-07-23
Filing date: 2019-07-23
Publication date: 2019-10-22

Abstract

The present invention relates to a kind of polygenes to recombinate Lactobacillus casei, preparation method and application, it is cloned by T-A and target gene is connected into pMD18-T respectively, conversion to DH5 α competent cell obtains pMD18-T-S1, pMD18-T-GtxA-N and pMD18-T-flfA, respectively with pMD18-T-S1, pMD18-T-GtxA-N and pMD18-T-flfA is that template is expanded, obtain three target fragments, three target fragments are connected as a bar segment, PCR amplification pMG36e is so that it becomes linearized vector, the segment is connected with linearized vector by seamless clone technology, and connection product is converted and obtains recombinant plasmid into XL1-Bule competent cell, its electrotransformation is entered Recombination Lactobacillus casei is obtained in Lactobacillus casei.

Description

A kind of polygenes recombination Lactobacillus casei, preparation method and application

Technical field

The invention belongs to microorganisms technical field, in particular to a kind of polygenes recombinates Lactobacillus casei, preparation method And application.

Background technique

Laying hen cyst of fallopian tube is always the important epidemic disease for endangering aviculture, and the disease is with caseous salpingitis, fallopian tubal Blocking is characterized, and often leads to the chicken egg productivity of egg-laying peak and quality of laying eggs declines rapidly, especially the high yield of current year life Hen is more common.Have been generally acknowledged that infectious bronchitis virus, duck source chicken bacillus (G.anatis), Egg Drop syndrome virus and clothing Substance etc. is to cause salpingitis and cyst of fallopian tube main pathogens, especially G.anatis.RTX toxin (Repeats in Toxin) it is present in a variety of Gram-negative bacterias, plays an important role in the pathogenic course to specific species, GtxA albumen It is duck source chicken bacillus RTX sample toxin, there is leucocytotoxicity and hemolytic activity, research shows that the N-terminal region of GtxA albumen has Good immunogenicity, and prove that recombinant protein GtxA-N can trigger effective immunoprotection and can be used as anti-G.anatis's processed Vaccine antigen.F17 sample pili is the important virulence factor of G.anatis, and can be used as that non-serotype relies on it is conservative potential Vaccine candidate albumen.The main infringement chicken respiratory system of IBV virus and digestion reproductive system, and it is believed that chicken salpingo tumour Generation it is related with chicken IBV virus early infection, meanwhile, tend to isolate IBV virus in the chicken body of G.anatis infection, subtract Egg syndrome virus and mycoplasma etc., therefore, IBV virus are also the important pathogen for causing laying hen cyst of fallopian tube.IBV virus base 4 kinds of structural proteins are encoded altogether because organizing, and wherein prominent (S) albumen of the fibre on outer virionic membrane needs 2 that are cracked into S1 and S2 upon translation Protein protomer could become the maturation protein for having biological activity, and Sl albumen is the major antigen of IBV virus.

Currently, G.anatis and IBV have been in global distribution, and it can cause defeated ovum after infecting laying hen researches show that the two Guan Yan and cyst of fallopian tube, therefore, to both disease Synthetical prevention can largely reduce laying hen cyst of fallopian tube and The economic loss of laying hen industry is effectively reduced in the generation of salpingitis.Vaccine immunity is the important means of prevention and control epidemic disease, develops peace Entirely, G.anatis and IBV combined vaccine efficiently, practical seems promising important.The selection of vaccine is in entire immunoprotection process In it is extremely important, be the important approach of infection through mucous membrane intrusion host, therefore disease can effectively be resisted by mucosa-immune The intrusion of substance.

Body mucosal immune system can be stimulated to generate local specific immune response for this reason, it may be necessary to develop one kind, to draw The vaccine of systemic immunity reaction is played, immunoprotection thus is carried out to poultry.

Summary of the invention

The purpose of the present invention is to provide one kind can prepare safe and efficient, practical G.anatis and IBV combined vaccine Polygenes recombinates Lactobacillus casei.

A kind of polygenes provided by the invention recombinates Lactobacillus casei, by recombinant plasmid pMG36e-S1-GtxA-flfA Electrotransformation to cheese lactic acid competent cell bacillus obtains；The polygenes recombination Lactobacillus casei is by recombinant plasmid PMG36e-S1-GtxA-flfA electrotransformation to cheese lactic acid competent cell bacillus obtains；The recombinant plasmid pMG36e-S1- GtxA-flfA is to be connected into fusion S1-GtxA-flfA between the site kPnI of linear plasmid pMG36e and the site SalI to obtain It arrives, the nucleotide sequence of the fusion S1-GtxA-flfA is as shown in SEQ ID NO.19.

Further, the Lactobacillus casei is Lb.casei CECT5276.

The present invention also provides the preparation methods of above-mentioned polygenes recombination Lactobacillus casei, include the following steps:

S1 designs specificity amplification primer 1, such as sequence table of upstream and downstream primer using IBV Jin-13 virus cDNA as template Shown in SEQ ID NO:1 and SEQ ID NO:2, carries out PCR amplification and obtain IBV virus S1 genetic fragment；Amplified fragments are recycled through glue It is attached afterwards with pMD18-T carrier, connection product is converted to bacillus coli DH 5 alpha competent cell, and carry out positive colony The identification of screening and recombination positive plasmid, obtains recombinant plasmid pMD18-T-S1；

Using G.anatis UMN179 full-length genome as template, specificity amplification primer 2, upstream and downstream primer such as sequence are designed Shown in table SEQ ID NO:3 and SEQ ID NO:4, carries out PCR amplification and obtain G.anatisGtxA genetic fragment；Amplified fragments are through glue It is attached after recycling with pMD18-T carrier, connection product is converted to bacillus coli DH 5 alpha competent cell, and carry out the positive The identification of colony screening and recombination positive plasmid, obtains recombinant plasmid pMD18-T-GtxA；

Using G.anatis full-length genome as template, specificity amplification primer 3, upstream and downstream primer such as sequence table SEQ are designed Shown in ID NO:5 and SEQ ID NO:6, carries out PCR amplification and obtain G.anatis flfA genetic fragment, amplified fragments are recycled through glue It is attached afterwards with pMD18-T carrier, connection product is converted to bacillus coli DH 5 alpha competent cell, and carry out positive colony The identification of screening and recombination positive plasmid, obtains recombinant plasmid pMD18-T-flfA.

S2 designs specificity amplification primer 4, such as sequence table of upstream and downstream primer with recombinant plasmid pMD18-T-S1 template Shown in SEQ ID NO:7 and SEQ ID NO:8, PCR amplification is carried out, identified result correctly carries out glue recycling afterwards, obtains purpose Segment 1；

With recombinant plasmid pMD18-T-GtxA template, specificity amplification primer 5, upstream and downstream primer such as sequence table SEQ are designed Shown in ID NO:9 and SEQ ID NO:10, PCR amplification is carried out, it is identified, glue recycling is carried out after as a result correct, obtains purpose piece Section 2；

With recombinant plasmid pMD18-T-flf template, specificity amplification primer 6, upstream and downstream primer such as sequence table SEQ are designed Shown in ID NO:11 and SEQ ID NO:12, PCR amplification is carried out, identified result correctly carries out glue recycling afterwards, obtains purpose piece Section 3；

S3 is once expanded target fragment 1, target fragment 2 and target fragment 3 by overlapping PCR method, one It is secondary amplification terminate after into PCR system be added specificity amplification primer 7, upstream and downstream primer such as sequence table SEQ ID NO:13 and Shown in SEQ ID NO:14, secondary amplification is carried out, correct amplified production recycling will be identified, obtains fusion S1-GtxA- flfA；

S4 designs specificity amplification primer 8, upstream and downstream primer such as sequence table SEQ ID using pMG36e plasmid as template Shown in NO:15 and SEQ ID NO:16, PCR amplification is carried out, obtains linear plasmid pMG36e；

Fusion S1-GtxA-flfA and linear plasmid pMG36e are attached by S5 by seamless clone technology, will Connection product is converted to Escherichia coli XL1-Bule competent cell, obtains recombinant plasmid pMG36e-S1-GtxA-flfA；

S6 mixes recombinant plasmid pMG36e-S1-GtxA-flfA with Lactobacillus casei competent cell, ice bath 5min, then high-voltage pulse electric shock converts, much genetic recombination Lactobacillus casei.

Further, the preparation method of above-mentioned polygenes recombination Lactobacillus casei, in S1, PCR system are as follows: 2 × Easy Taq Super PCR Master Mix 25ul, upstream primer 1ul, downstream primer 1ul, template DNA 21ul；

PCR program are as follows:

95 DEG C, 5min；

30 circulations:

95 DEG C, 30s,

56 DEG C, 30s,

72 DEG C, primer 1 expands 60s/ primer 2 amplification 90s/ primer 3 and expands 30s；

72℃,10min。

Compared with prior art, beneficial effects of the present invention: the present invention successfully constructs one plant of coexpression IBV-S1 and Ya Yuan Chicken bacillus FlfA, gtxA recombinate Lactobacillus casei, are a kind of mucous membrane microorganisms in conjunction with duck source chicken bacillus, are mainly colonized in and exhale Road, alimentary canal and urogenital system this feature are inhaled, in preparation for the live vector epidemic disease of coexpression S1-GtxA-FlfA albumen Select lactic acid bacteria as the carrier of present antigen when seedling, it is not only safe and non-toxic in this way, but also body intestinal mucosa can be induced to produce Raw immune response, irritating body generates whole body specific immune response, to play a protective role.

Detailed description of the invention

Fig. 1 is PCR amplification result figure (the M:DNA Marker 250Ladder of 1 three kinds of genes of the embodiment of the present invention；1: The PCR product of IBV S1 gene；2:G.anatis GtxA gene PCR product；3:G.anatis flfA gene PCR product).

The PCR that Fig. 2 is 1 recombinant plasmid pMD18-T-S1 of the embodiment of the present invention identifies (M:DNA Marker250Ladder； The PCR product of 1:IBV S1 gene；2: plasmid pMD18-T-S1).

The PCR that Fig. 3 is 1 recombinant plasmid pMD18-T-GtxA of the embodiment of the present invention identifies (M:DNA Marker 250Ladder；The PCR product of 1:G.anatis GtxA gene；2: plasmid pMD18-T-GtxA).

Fig. 4 is the PCR identification of M of 1 recombinant plasmid pMD18-T-GtxA of the embodiment of the present invention: DNA Marker 250Ladder；The PCR product of 1:G.anatis flfA gene；2: plasmid pMD18-T-flfA).

Fig. 5 is PCR amplification result (the M:DNA Marker DL2000 of 1 target fragment of the embodiment of the present invention；1:IBV S1 The PCR product of gene；2:G.anatis GtxA gene PCR product；3:G.anatis flfA).

Fig. 6 is concatenated target gene over-lap PCR amplification (the M:DNA Marker DL of the embodiment of the present invention 1 15000；1: fusion segment S1-GtxA-flfA).

Fig. 7 is PCR amplification result (the M:DNA Marker DL15000 of 1 plasmid pMG36e of the embodiment of the present invention；1: plasmid pMG36e)。

Fig. 8 is PCR amplification result (M: the λ-Hind of 1 recombinant plasmid pMG36e-S1-GtxA-flfA of the embodiment of the present invention III digest DNA Marker；1-4: recombinant plasmid pMG36e-S1-GtxA-flfA).

Fig. 9 is identification (the M:DNA Marker DL 15000 of 2 recombinant bacterium of the embodiment of the present invention；1-5:L.casei 5276 In plasmid recombinant plasmid pMG36e-S1-GtxA-flfA；6: negative control).

Figure 10 is the growth curve of the recombinant bacterium of the embodiment of the present invention 3.

Figure 11 is that the external genetic stability of recombination Lactobacillus casei of the embodiment of the present invention 4 analyzes result (M:DNA Mareker 250Ladder；PCR product of the 1-10:S1-GtxA-flfA gene in 1-20 generation recombination Lactobacillus casei；11: Negative control)

Specific embodiment

With reference to the accompanying drawing and the specific embodiment of the present invention is described in detail in specific embodiment, but answers When understanding that the protection scope of the present invention is not limited by the specific implementation manner, operating process of the present invention is such as without special theory Bright is conventional method, and being related to reagent is to be commercially available on the market.

IBVJin-13 virus used in the present invention is recorded in " " Agricultural University Of Anhui's journal " 01 phase in 2012, paper name Claim " Jin13 plants of avian infectious bronchitis virus of separation and identification " " in, applicant's guarantee is from applying for mesh to public affairs in 20 years Crowd provides biomaterial.

Embodiment 1

Polygenes of the invention recombinates Lactobacillus casei, extremely by recombinant plasmid pMG36e-S1-GtxA-flfA electrotransformation Cheese lactic acid competent cell bacillus obtains；The recombinant plasmid pMG36e-S1-GtxA-flfA is by fusion S1- GtxA-flfA is connected between the site kPnI of linear plasmid pMG36e and the site SalI, the fusion S1-GtxA-flfA Nucleotide sequence as shown in SEQ ID NO.19.

A kind of preparation method of polygenes recombination Lactobacillus casei, its step are as follows:

According to GenBank IBV Jin-13 virus S1 gene, GtxA, flfA gene of G.anatis UMN179 and shuttle Expression vector pMG36e sequence, using primer-design software Premier 5.0 design multipair specific primer for construct recombination Plasmid pMG36e-S1/GtxA/flfA, primer are synthesized by giving birth to work bioengineering (Zhengzhou) Co., Ltd.

Using G.anatis full-length genome as template, specificity amplification primer 2, upstream and downstream primer such as sequence table SEQ are designed Shown in ID NO:3 and SEQ ID NO:4, carries out PCR amplification and obtain G.anatis GtxA genetic fragment；Amplified fragments are recycled through glue It is attached afterwards with pMD18-T carrier, connection product is converted to bacillus coli DH 5 alpha competent cell, and carry out positive colony The identification of screening and recombination positive plasmid, obtains recombinant plasmid pMD18-T-GtxA；

Using G.anatis full-length genome as template, specificity amplification primer 3, upstream and downstream primer such as sequence table SEQ are designed Shown in ID NO:5 and SEQ ID NO:6, carries out PCR amplification and obtain G.anatis flfA genetic fragment, obtain recombinant plasmid pMD18-T-flfA。

The system and amplification program that three PCR react in S1 are as shown in table 1,2:

The PCR reaction system of 1 S1 of table

2 PCR amplification program of table

Pcr amplification product in 1%TAE agarose gel electrophoresis identify, as a result as shown in Figure 1 (M:DNAMarker 250Ladder；The PCR product of 1:IBV S1 gene；2:G.anatis GtxA gene PCR product；G.anatis flfA gene PCR product), and according to DNA gel electrophoresis kit specification, purification and recovery specific band.

Three glue recovery product fS1 genes, GtxA gene and flfA gene, then connect with carrier T respectively in S1, connection System such as table 3:

3 linked system of table

Three linked system mixtures are placed in 16 DEG C of link slot and carry out staying overnight connection, then by three connection products It is converted respectively to bacillus coli DH 5 alpha competent cell, specific steps are as follows:

(1) 10 μ L connection products and 100 μ L bacillus coli DH 5 alphas are mixed by state cell, ice bath 30min；

(2) in 42 DEG C of water-bath heat shock 90s, ice bath 5min in mixture of ice and water is moved to；

(3) the LB culture medium of 1mL, 200r/min, 37 DEG C of culture 1h are added into EP pipe；

(4) bacterium solution 10000rpm, 4 DEG C of centrifugation 5min after cultivating use remaining bacterium solution in EP pipe after abandoning 900 μ L supernatants Liquid-transfering gun mixing takes appropriate bacterium solution to be coated in the LB solid medium containing 100 μ g/mL Amp, and 37 DEG C of stationary cultures are stayed overnight, then Carry out positive colony screening.

The recombination positive plasmid identification system of three connection products is essentially identical, and difference is primer difference, corresponding to draw Object is respectively primer 1, primer 2 and primer 3, it is specific as shown in table 4:

4 recombinant plasmid PCR identification system of table

PCR amplification condition is identified in 1%TAE agarose gel electrophoresis with its amplified production of table 2, is expanded by PCR Increasing and identify, obtains correct target fragment, qualification result is as shown in Figure 2,3, 4, position size respectively about 1608bp, 2847bp, 549bp, sequencing result are correct.

Three PCR amplification systems are essentially identical in S2, and difference is primer difference, and primer is respectively primer 4,5 and of primer Primer 6, PCR reaction system such as table 5:

The PCR reaction system of 5 S2 of table

Each pcr amplification product is identified that qualification result is as schemed by amplification condition in 1%TAE agarose gel electrophoresis Correct through sequencing result shown in 5, phenomena such as mispairing does not occur for base sequence, carries out glue after as a result correct and recycles target fragment ,- 20 DEG C save backup.

S3 is once expanded target fragment 1, target fragment 2 and target fragment 3 by overlapping PCR method, one Specificity amplification primer 7, each 2uL of upstream and downstream primer, upstream and downstream primer such as sequence are added into PCR system after terminating for secondary amplification Shown in table SEQ ID NO:13 and SEQ ID NO:14, secondary amplification is carried out, correct amplified production recycling will be identified, is melted Close gene S1-GtxA-flfA；

The reaction system and reaction condition once expanded such as table 6,7:

Table amplification reaction system of 6 over-lap PCR

Table amplification condition of 7 over-lap PCR

The reaction condition of secondary amplification such as table 8:

Table amplification condition of 8 over-lap PCR

It is identified through 1%TAE agarose gel electrophoresis, qualification result at about 5052bp as shown in fig. 6, there is a specificity Purpose band, it is consistent with expected results to pass through, PCR product purification kit recycle -20 DEG C save backup.

PCR reaction system and reaction condition such as table 9,10:

The PCR reaction system of the amplification of table 9 plasmid pMG36e

10 PCR amplification condition of table

It is identified through 1%TAE agarose gel electrophoresis, qualification result is as shown in fig. 7, obtain the specific item of about 3600bp Band, as a result correctly.

It after 1 μ L DpnI enzymic digestion is added into PCR product, is recycled using PCR product purification kit, -20 DEG C of guarantors It deposits spare.

Fusion S1-GtxA-flfA and linear plasmid pMG36e are attached by seamless clone technology, are obtained by S5 To recombinant plasmid pMG36e-S1-GtxA-flfA；Reaction system and response procedures such as table 11,12:

The seamless cloning reaction system of table 11

12 PCR amplification condition of table

After the identification of 1%TAE agarose gel electrophoresis, -20 DEG C are recycled through PCR product purification kit and is saved backup.

Seamless cloned sequence is gone into Escherichia coli XLI-Blue competent cell, operating procedure is as follows:

(1) 10 μ L connection products and 100 μ L Escherichia coli XLI-Blue competent cells are mixed, ice bath 30min；

The step of positive colony screens are as follows: the single bacterium of the doubtful positive recombinant plasmid of picking is fallen within containing final concentration of 300 μ g/mL In the MRS culture medium of erythromycin, 37 DEG C of stationary cultures for 24 hours after, design specificity amplification primer 6, such as sequence table of upstream and downstream primer SEQ ID NO:17 and SEQ ID NO:18 carries out PCR identification, PCR identification system and reaction condition such as table 13,14:

13 recombinant plasmid pMG36e-S1-GtxA-flfA PCR identification system of table

14 PCR amplification condition of table

PCR product is identified in 1%TAE agarose gel electrophoresis, qualification result is as shown in figure 8, obtain about 5376bp purpose band, through being sequenced, as a result correctly.

Wherein, the preparation of Lactobacillus casei competent cell includes the following steps:

Lactobacillus casei (Lb.casei CECT5276) strain that -80 DEG C are saved, the MRS for lining non-resistant are solid On body culture medium, 37 DEG C are stood for 24 hours, and picking single bacterium is fallen in the MRS culture medium of 5mL non-resistant, 37 DEG C of standing 18h；Take 2mL bacterium Liquid is in 100mLMRS culture medium, 37 DEG C of cultures to OD₆₀₀It is 0.3~0.5.After collecting thallus ice bath 5min, 4 DEG C, 10000rpm is centrifuged 15min, abandons supernatant, and 10% glycerol washing thalline being pre-chilled in equal volume is added, and washes repeatedly 3 times, is added 1mL is pre-chilled 10% glycerol and is resuspended.100 μ L of the competent cell prepared is taken to be sub-packed in 1.5mLEP pipe, -80 DEG C save backup.

The step of electrotransformation are as follows:

Taking 10 μ L concentration is the matter recombinant plasmid pMG36e-S1-GtxA-flfA of 1 μ g/mL, with the above-mentioned cheese prepared For Bacillus acidi lactici by being mixed in state cell EP pipe (plasmid solution volume is no more than competence solution 1:10), ice bath 5min will Mixed liquor is added in the sterile 2mm electricity revolving cup of pre-cooling, high-voltage pulse electric shock conversion.Conversion condition: electric field strength 2000V, 200 Ω of resistance, 25 μ F of capacitor.The SMRS fluid nutrient medium of 890 μ L is added in electric shock into electric revolving cup after finishing, moved after mixing Into sterile EP tube, 37 DEG C of standing 3h.4 DEG C, 10000rpm, it is centrifuged 15min, 800 μ L supernatants are discarded, by remaining liq and bacterium Body mixes, and is coated in the MRS solid medium containing 5 μ g/mL erythromycin, and observation as a result, use simultaneously after 37 DEG C of stationary culture 48h PMG36e empty carrier conversion Lactobacillus casei compares.Doubtful recombinant lactic acid bacteria clone on picking plate, is connected to 5mL containing 5 μ In the MRS culture medium of g/mL erythromycin, 37 DEG C of stationary culture 48h.

Embodiment 2

Recombinate the identification of Lactobacillus casei

Picking positive single bacterium is fallen in the MRS solid medium containing final concentration of 5 μ g/mL erythromycin, and 37 DEG C stand overnight Culture, and PCR identification is carried out to bacterium solution.PCR identification system and reaction condition such as table 15,16:

Table 15 recombinates Lactobacillus casei PCR identification system

PCR amplification condition identifies in 1%TAE agarose gel electrophoresis with table 14, by PCR product, qualification result As shown in figure 9, obtaining the purpose band of about 5376bp, position is correct.

Embodiment 3

Recombinate the growth conditions analysis of Lactobacillus casei

Recombinant bacterium is activated respectively with corresponding empty bacterium, recombinant bacterium is connected to the MRS containing 5mL containing 5 μ g/mL erythromycin and trains It supports in base, empty bacterium is connected in non-resistant MRS culture medium, after 37 DEG C stand for 24 hours, then takes 1mL bacterium solution in being connected to 100mL MRS respectively In culture medium, use MRS culture medium as blank control；37 DEG C of stationary cultures sample every 2h since 0h, measure culture solution OD₆₀₀Value continuously detects 14h, draws growth curve.The results are shown in Figure 10, the recombination cheese lactic acid bar speed of growth and empty cheese Bacillus acidi lactici is compared, and without significant difference between them, the insertion of illustration purpose gene has no influence to host strain.

Embodiment 4

Genetic stability analysis of the target gene in recombination Lactobacillus casei

It chooses recombinant bacterium single bacterium to fall in MRS culture medium of the 5mL containing 1 μ g/mL erythromycin, 37 DEG C of standings are trained under conditions of for 24 hours It supported for 20 generations, extracts bacterial genomes every a generation, for No. 9 specific primers to PCR identification is carried out, reaction system and reaction condition are same 2.6.3, PCR product is observed in 1%TAE agarose gel electrophoresis as a result, result is as shown in figure 11, in each generation recombinant bacterium The purpose band of length about 5376bp can be amplified, it was demonstrated that fusion S1-GtxA-flfA can stablize in Lactobacillus casei Heredity.

It should be noted that the step method used in claims of the present invention is same as the previously described embodiments, in order to anti- It only repeats, description of the invention preferred embodiment, once a person skilled in the art knows basic creative general It reads, then additional changes and modifications can be made to these embodiments.So it includes preferred real that the following claims are intended to be interpreted as It applies example and falls into all change and modification of the scope of the invention.

Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies Within, then the present invention is also intended to include these modifications and variations.

Sequence table

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cttttctata atttaacagt tagtgtagct aagtacccta cttttaaatc atttcagtgt 480

gttaataatt taacatccgt atatttaaat ggtgatcttg tttacacctc taatgagacc 540

acagatgtta catctgcagg tgtttatttt aaagctggtg gacctataac ttataaagtt 600

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aatagtgtta atactacttt tacgttacac aatttcactt ttcataatga gactggcgcc 840

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atgtatggat cttatcaccc aagttgtaat tttagactag aaactattaa taatggcttg 1020

tggtttaatt cactttcagt ttcaattgct tacggtcctc ttcaaggtgg ttgcaagcaa 1080

tctgtcttta gtggtagagc aacttgttgt tatgcttatt catatggagg tccttcgctg 1140

tgtaaaggtg tttattcagg tgagttagat cttaattttg aatgtggact gttagtttat 1200

gttactaaga gcggtggctc tcgtatacaa acagccactg aaccgccagt tataactcga 1260

cacaattata ataatattac tttaaatact tgtgttgatt ataatatata tggcagaact 1320

ggccaaggtt ttattactaa tgtaaccgac tcagctgtta gttataatta tctagcagac 1380

gcaggtttgg ctattttaga tacatctggt tccatagaca tctttgttgt acaaggtgaa 1440

tatggtctta cttattataa ggttaaccct tgcgaagatg tcaaccagca gtttgtagtt 1500

tctggtggta aattagtagg tattcttact tcacgtaatg agactggttc tcagcttctt 1560

gagaaccagt tttacattaa aatcactaat ggaacacgtc gttttagaat gctttcatta 1620

aaagaaaaag taactggaat agattttgat gcaatcaaag ataaagtcgt ttcattaaaa 1680

aacacggttt caaatattga ttttaatctg gttaaagaag atatttcttc tttaaaaagc 1740

aatgcgttat ccatcgcggc atcagatttt aaaaataaac cggtgttatt caaagactct 1800

ttagacttac ttactgatgc tacaaataca ctcagaaaga ttaccaatca aatgtcatca 1860

attagcgaaa tttctaataa gtcattagat ttgctggatt ctctttttga ggctgccaaa 1920

gatattgtaa acattgccta ttcaaaaggt ggtgtcgaaa ttactaagtc tgcgacagaa 1980

ttagcggcaa aagcggcatt aattgttgat aaaagtatca tattagcaaa taaagataat 2040

acaattagtg aagctgttta tcattctatt aacaactcat tacaaaatat tcaaaaaaca 2100

gctatcaata ttgctacaca ttcacataat gaagataaag ctgaaattgc taaagcctct 2160

tttgagctgt tatctcaagt tagtgatgtt atcagtaatg cgttaaaaaa ttcaggtgat 2220

ataggtatcg aatcacaact cttagccgat attaatcagt tttctcattc tattttgaac 2280

acagctaaaa cagttactga tatagctact atggatatga atgataaaac ctcaatcgct 2340

aaaaatagca tttcattaat agccaaagtg aatgatgtta tttccgatat tctagtaatg 2400

acggataaag acaccgaatt attaaatgca attcataatg ttactgcgaa aaatctacag 2460

aatatcgaag agagtgcggt caatcttgca aatgctgatg tgctgtctca agaaggcaaa 2520

gtcagtattg ccattaattc tttaacttta atatcacaaa ccaacaaaat tgttgcgcaa 2580

gtgctaaatg aagctaattt aagcactgat aaaacccaat ttgttggcga attaaccgat 2640

gtattattga ataccgcaaa aagtattaca ttgttagcta ccggtaataa tgcgacaaca 2700

gcaggaaagg aacagctggc agttgcctca accaatctta ttggtaacgt gaatgatctc 2760

attcaatcaa ttaccagctt taaaggcaaa gaagatattg gtaacgcttt acacagtgcg 2820

gtggacggac aattatcaca aatcaaacaa cttgcggtcg cgttatcaaa cagtaatctt 2880

gattcttcac aaggtaaaac tgcaatagcc atcacctctt tcggcttgat tgcacaagca 2940

aataatatta tcaataaatt cttggataat atgagtttaa gtactaatgt gagtaaatcg 3000

gttcatagtt tgactaattc agcgctagat gcagccaaaa ttctcacaaa cgtagtacaa 3060

gtagatgcta ataacaatca aggaaaggtc gtgattgcca atagttcatt agaactttct 3120

aaaacagcaa gtgatattgt gtctactgtg ttaaaaagca catctatttc aacacaacat 3180

attgatataa ttcataatgc agtaaataaa acattaacag aaatgaaaga tagtgcggta 3240

gcaatagcac ttgcttcatc tgaaaataat agcgctgaaa ttgcaacgca ttcattaagt 3300

ctgttatccg atgcaagtaa tatgttgaaa gatattatgc aaggaatgag ccctaataat 3360

gtcattgctc cgaaaacatt agaattattt aactcactat ttgcgacagc tcaaaatatc 3420

gttcaattag ctgacgcaaa atcttcagaa aacattgcta aagctagtgt tgatttggta 3480

caaagcgcaa cgattatcct caataacgta ttaacgttgg ctaacgttga ttcttcttta 3540

agtaaagctt ttcatcaatc atttgatgct tcagtttctc aaattaaaga ggtagcagct 3600

caattagcta ccgcgtcttc tgcctctaat aaagctgaga ttgcaaaact ctcttttgat 3660

tttattagtc aagtaagtga tttagcgacc aacaccttaa caacagcgaa aaccggatta 3720

gatagcacgc tgctgaataa cgttaacggt ctttctcatt ccgtcttaaa tgcagcaaaa 3780

tcagtaaccg atattattgt gagtgataac ccagcgaata ccgccagttt atccgtttct 3840

ttggtgaata atgccaatga aattgtttca aatatcttaa ccttatccgg aaaacaaaat 3900

acgctctcca cggcagtaca cgatataacc gctaaacatt tagcgccgat tgagaaaata 3960

gcaattaacc ttgcgaatgc cgataactca agcagtgatg gaaaagttgc tattgcgtta 4020

aactcattaa cattgattgc acaaagtaac catttaatcg aagaagtatt aaaagaggct 4080

aaattagata atgcgaagag cgcctttgct cacaatttaa cggatttagt attagatacc 4140

gccaaaacaa tcacggcatt agcatcagcg gataccagta aagtagacgg caagcagcag 4200

attgcctctg catcaacaca tttagtcgga caaattaatg agattgtcaa atcaatcacg 4260

acaataacca attcagaaac gaaagtcggc aatgccgcat atcaagcgtt aaaaacacat 4320

ttagagcagg tagaaacaat tgcggttaaa cttgccgccg ccaatgcatc aacagcggaa 4380

ggcagaacag aaattgcgat tgaatctttc aatttaatcg caaaaaccaa tggcataatg 4440

accgatttcc taaatatgaa aaaattgctt ttaacaactt taattgccgt tggtttgggc 4500

ttatcagcac aaggtgcatt tgcggatgat cctagtgcag ctaattcaac aaatggcggt 4560

cttattacaa tcactggtaa agtaacagat accacttgtt taattaatgg aaaagaaaat 4620

ggcgatttga cagtacaatt accaccagtt tctacaaaag cgctagctac tcagggcgca 4680

acaacaggtg atacagcatt tacaattgaa ttaagtggtt gtcaatctgc aacagataaa 4740

cttgcaaaaa aagcagcagc attcttcgtg aatgaatcag ataaagtaac agctgatggt 4800

aagttactaa ataaacctgc aactcctggt gatgcagctc aaaatgtggt ggtgcaatta 4860

cttcacggtg atgatactcc aattgatatt acaaaacctt atgcggaaca aactaaagag 4920

gctcaaaaag ctggaattga ttcaagcaag aaaacagcca cattgaaata taaagctcgt 4980

tactatgcaa ctaatccggc aggagcgggt gaggtgaaat ctaccgtgaa ctatactatc 5040

gcatacgaat aaggtacccc g 5061

Claims

1. a kind of polygenes recombinates Lactobacillus casei, which is characterized in that the polygenes recombination Lactobacillus casei is by recombinating Plasmid pMG36e-S1-GtxA-flfA electrotransformation to cheese lactic acid competent cell bacillus obtains；The recombinant plasmid pMG36e- S1-GtxA-flfA be fusion S1-GtxA-flfA is connected into linear plasmid pMG36e the site kPnI and the site SalI it Between obtain, the nucleotide sequence of the fusion S1-GtxA-flfA is as shown in SEQ ID NO.19.

2. a kind of polygenes as described in claim 1 recombinates Lactobacillus casei, which is characterized in that the Lactobacillus casei For Lb.caseiCECT5276.

3. the preparation method of polygenes recombination Lactobacillus casei as described in claim 1, which is characterized in that including following step It is rapid:

S1 designs specificity amplification primer 1, upstream and downstream primer such as sequence table SEQ using IBV Jin-13 virus cDNA as template Shown in ID NO:1 and SEQ ID NO:2, carries out PCR amplification and obtain IBV virus S1 genetic fragment；Amplified fragments after glue recycles with PMD18-T carrier is attached, and connection product is converted to bacillus coli DH 5 alpha competent cell, obtains recombinant plasmid pMD18- T-S1；

Using G.anatis full-length genome as template, specificity amplification primer 2, upstream and downstream primer such as sequence table SEQ ID are designed Shown in NO:3 and SEQ ID NO:4, carries out PCR amplification and obtain G.anatis GtxA genetic fragment；Amplified fragments after glue recycles with PMD18-T carrier is attached, and connection product is converted to bacillus coli DH 5 alpha competent cell, obtains recombinant plasmid pMD18- T-GtxA；

Using G.anatis full-length genome as template, specificity amplification primer 3, upstream and downstream primer such as sequence table SEQ ID are designed Shown in NO:5 and SEQ ID NO:6, carry out PCR amplification and obtain G.anatis flfA genetic fragment, amplified fragments after glue recycles with PMD18-T carrier is attached, and connection product is converted to bacillus coli DH 5 alpha competent cell, obtains recombinant plasmid pMD18- T-flfA。

S2 designs specificity amplification primer 4, upstream and downstream primer such as sequence table SEQ ID with recombinant plasmid pMD18-T-S1 template Shown in NO:7 and SEQ ID NO:8, PCR amplification is carried out, identified result correctly carries out glue recycling afterwards, obtains target fragment 1；

With recombinant plasmid pMD18-T-GtxA template, specificity amplification primer 5, upstream and downstream primer such as sequence table SEQ ID are designed Shown in NO:9 and SEQ ID NO:10, PCR amplification is carried out, it is identified, glue recycling is carried out after as a result correct, obtains target fragment 2；

With recombinant plasmid pMD18-T-flf template, specificity amplification primer 6, upstream and downstream primer such as sequence table SEQ ID are designed Shown in NO:11 and SEQ ID NO:12, PCR amplification is carried out, identified result correctly carries out glue recycling afterwards, obtains target fragment 3；

S3 is once expanded target fragment 1, target fragment 2 and target fragment 3 by overlapping PCR method, primary to expand Specificity amplification primer 7, upstream and downstream primer such as sequence table SEQ ID NO:13 and SEQ are added into PCR system after terminating for increasing Shown in ID NO:14, secondary amplification is carried out, correct amplified production recycling will be identified, obtains fusion S1-GtxA-flfA；

S4 designs specificity amplification primer 8, upstream and downstream primer such as sequence table SEQ ID NO:15 using pMG36e plasmid as template With shown in SEQ ID NO:16, PCR amplification is carried out, obtains linear plasmid pMG36e；

Fusion S1-GtxA-flfA and linear plasmid pMG36e are attached by seamless clone technology, will be connected by S5 Product is converted to Escherichia coli XL1-Bule competent cell, obtains recombinant plasmid pMG36e-S1-GtxA-flfA；

S6 mixes recombinant plasmid pMG36e-S1-GtxA-flfA with Lactobacillus casei competent cell, ice bath 5min, so High-voltage pulse electric shock converts afterwards, much genetic recombination Lactobacillus casei.

4. the preparation method of polygenes recombination Lactobacillus casei as claimed in claim 3, which is characterized in that in S1, PCR body System are as follows: 2 × Easy Taq Super PCR Master Mix 25ul, upstream primer 1ul, downstream primer 1ul, template DNA21ul；

PCR program are as follows:

95 DEG C, 5min；

30 circulations:

95 DEG C, 30s,

56 DEG C, 30s,

72℃,10min。