CN104357461A - Method for establishing S gene of piglet diarrhea virus variant part and prokaryotic expression vector of S gene - Google Patents
Method for establishing S gene of piglet diarrhea virus variant part and prokaryotic expression vector of S gene Download PDFInfo
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- CN104357461A CN104357461A CN201410710763.5A CN201410710763A CN104357461A CN 104357461 A CN104357461 A CN 104357461A CN 201410710763 A CN201410710763 A CN 201410710763A CN 104357461 A CN104357461 A CN 104357461A
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Abstract
The invention belongs to the field of genetic engineering, and in particular relates to a method for establishing an S gene of a piglet diarrhea virus variant part and a prokaryotic expression vector of the S gene. The method comprises the following steps: (a) by taking F1 and R1 as primers (F1 is 5'-GAATTCGATGTCACCAGGTGCT-3' and R1 is 5'-GTCGACTTAATTAAATGATGG-3'), performing PCR (Polymerase Chain Reaction) amplification, thereby obtaining a gene sequence as shown in SEQ ID NO.1, of the S gene of an encoded piglet diarrhea virus variant part; (b) connecting the S gene sequence of the encoded piglet diarrhea virus variant part to a pET32a carrier, thereby obtaining a recombinant expression carrier pET32a-bs1; and (c) transferring the recombinant expression carrier pET32a-bs1plsmaid into an escherichia coli BL21 strain, oscillating at the speed of 250rpm to culture till the logarithmic phase OD600 is 0.6-0.7, adding 1mmol/L of IPTG, and inducing target expression protein for 6 hours at 37 DEG C, namely completing the establishment of the prokaryotic expression vector of the S gene of the piglet diarrhea virus variant part.
Description
Technical field
The invention belongs to genetically engineered field, be specifically related to the construction process of a kind of grice diarrhoea virus variant part S gene and prokaryotic expression carrier thereof.
Background technology
Porcine epizootic diarrhea is, the high degree in contact infectious intestinal disease acute with the one of suffering from diarrhoea, vomitting, dewatering and sucking piglets height lethality rate being principal character caused by virus.Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), only has a serotype, belongs to coronaviridae coronavirus genus, can infect the pig at various age and all fall ill.
PEDV clinical manifestation is watery diarrhea, and with vomiting; All age in days pigs all can occur, sucking piglets within 10 ages in days of being wherein born, usually after maintenance is suffered from diarrhoea 2-4 days, and dehydration mortality (50%-100%).The pig farm almost whole nest morbidity in some places, in watery diarrhea, sickness rate is high, and case fatality rate is high, and not dead piglet becomes weak son and cad pig.The sickness rate of the sucking piglets that age in days is larger and case fatality rate more relatively low.PEDV is to infect sucking pig, to cause heavy losses for feature; Since about 2000 Korea S are very popular, surrounding area all has breaks out, and China is in endemic conditions state always; PEDV, after first partial area break out, fails timely and effective control, after spreading gradually, occurs being very popular.
Since PEDV finds, scholars uses multiple method to attempt making virus adapt to cell, carries out cultured and amplified in vitro.Sweden Hoffman in 1988 etc. add pancreatin first in the nutrient solution of Vero passage cell, are successfully obtained the cell cultures of virus by blind passage continuous on cell.Japan and Korea S also have cultivation successfully report in succession subsequently.But the Vero cell of different sources, different strain and culture condition are all the important factors affecting virus purification.The Cell culture invitro of PEDV adapts to difficulty greatly, and Reproduction Conditions is complicated, and virus reproductive titer in cell is low, and cytopathy is not obvious, is an important technology bottleneck of PEDV in-vitro multiplication process always.Being separated to this virus of a strain needs the time longer, and workload is large, and the development of related vaccines is also affected and restriction.
At present, although commercialization PEDV vaccine generally uses, have the deactivation vaccine of multiple producer and Attenuate vaccine selective, immune effect is always undesirable, and this disease still has popular in China, and causes serious financial loss.By the analysis to virus, the structural protein of Porcine epidemic diarrhea virus are similar to other coronavirus, and prominent (spike) albumen of glycosyl chemical fibre is major structural protein.There is the property inserted sudden change in the wild poison of 2010PED, this region is the territory, antigen determination region of virus in spike glycoprotein gene region.The vaccine that before vaccine prepared by classical strains CV777 and variation prepared by strain (being in same evolutionary branching with classical strains), after virus S protein morphs, vaccine therapy effect is had a greatly reduced quality, and result for the treatment of is not obvious.In order to effectively treat the grice diarrhoea virus PEDV made a variation, develop a kind of effective PEDV vaccine newly very necessary.
Summary of the invention
Object of the present invention: (1) provides a kind of grice diarrhoea virus variant part S gene; (2) a kind of grice diarrhoea virus variant part S construction process of prokaryotic expression vector is provided; (3) expression method of a kind of grice diarrhoea virus variant part S gene in intestinal bacteria.
Technical scheme of the present invention: (1) a kind of grice diarrhoea virus variant part S gene, described S gene is made up of the nucleotide sequence shown in SEQ ID NO.1 in sequence table.(2) construction process for grice diarrhoea virus variant part S prokaryotic expression vector, is characterized in that: this construction process is made up of following steps:
(a) with F1 and R1 for primer, F1:5 '-GAATTCGATGTCACCAGGTGCT-3 '; R1:5 '-GTCGACTTAATTAAATGATGG-3 '; PCR reaction amplification is utilized to obtain the gene order of the coding grice diarrhoea virus variant part S gene as shown in SEQ ID NO.1; Described PCR reaction system is: 2 × Ex Taq PCR premix 10 μ l, cDNA 2 μ l, primer mixture 1 μ l, distilled water 7 μ l; Reaction parameter is divided into 3 steps, and the 1st step is 95 DEG C of 5min, and the 2nd step is 95 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 2min, 35 circulations, and the 3rd step is 72 DEG C and extends 10min;
B the grice diarrhoea variant part S gene order of above-mentioned coding is connected on pET32a carrier by (), obtain recombinant expression vector pET32a-bs1;
C recombinant expression vector pET32a-bs1 plasmid is proceeded to e. coli strain bl21 by (), 250rpm concussion is cultured to logarithmic phase OD
600=0.6 ~ 0.7, add 1mmol/L IPTG, 37 DEG C of induction 6h express the structure of target protein and grice diarrhoea virus variant part S prokaryotic expression vector, complete.
(3) expression method of a kind of grice diarrhoea virus variant part S gene in intestinal bacteria, this expression method has the following steps composition: the e. coli bl21 bacterium colony described in (1) picking claim 2, access contains antibiotic LB substratum, overnight incubation; (2) getting overnight culture transfers containing in antibiotic fresh LB nutrient solution, and 250rpm concussion is cultured to logarithmic phase OD
600=0.6 ~ 0.7; (3) in culture, add the IPTG that concentration is 1mmol/L, 37 DEG C, after abduction delivering 6h, 8000rpm, 20min centrifugal treating collects the coli somatic precipitation containing restructuring grice diarrhoea virus variant part S gene.
Described plasmid vector is containing SEQ ID NO.1 gene, is become by insertion pET32a vector construction.Described recombinant bacterial strain is obtained by above-mentioned plasmid vector transform both prokaryotic host cell strain.The prokaryotic host cell strain of this recombinant bacterial strain is intestinal bacteria.Described intestinal bacteria are e. coli bl21 (DE3).Above-mentioned intestinal bacteria are cultivated under suitable conditions in substratum.
The present invention constructs grice diarrhoea virus variant part S prokaryotic expression vector.Synthesize grice diarrhoea variant part S gene order by application round pcr, this gene order has been cloned in pET32a, obtains recombinant expression vector pET32a-bs1, then pass through CaCl
2transformation of E. coli, go out the higher positive colony transformant bs1 of expression amount according to growing state rapid screening, this expressing protein can be produced by Escherichia coli fermentation at present, has a good application prospect.
Beneficial effect: S protein is the important feature albumen at PEDV, can induce body to produce neutralizing antibody, is the main purpose gene of research PEDV recombinant vaccine.The present invention successfully constructs a strain recombined pronucleus expression albumen, can stably express PEDV part S gene protein.Prove that recombinant protein has reactionogenicity by western-blotting test; Piglet immunological test confirms, immune stimulating body creates the specific antibody for piglet epidemic diarrhea virus variant part S protein, and antibody Neutralizing titer reaches >=1:32, and constructed recombinant protein has immunogenicity.Part S gene constructed by the present invention comprises the insertion Mutational part that spike glycoprotein gene region occurs, for important foundation has been established in the research of PEDV novel variant strain gene engineering vaccine.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the present invention is further illustrated.
Fig. 1 is structure schema of the present invention;
The expression product electrophorogram of Fig. 2 recombinant protein in E.coli BL21.Wherein, band 1:pET-32a empty carrier induction; Band 2: recombinant expression vector pET-32a-bs1 does not induce; Band 3: recombinant expression vector pET-32a bs1 induces 4h; Band 4: recombinant expression vector pET-32a bs1 induces 5h; Band 5: recombinant expression vector pET-32a bs1 induces 6h; Band 6: recombinant expression vector pET-32a bs1 induces 7h; M: standard protein Marker
Embodiment
The acquisition of embodiment 1. grice diarrhoea virus variant part S gene order, according to the gene order of grice diarrhoea virus variant, designs following primer: F1:5 '-GAATTCGATGTCACCAGGTGCT-3 '; R1:5 '-GTCGACTTAATTAAATGATGG-3 ';
Utilize PCR to react amplification coding diarrhea virus variant part S gene order, PCR reaction system is (20 μ l): 2 × Ex TaqPCR premix 10 μ l, cDNA 2 μ l, primer mixture 1 μ l, distilled water 7 μ l.Reaction parameter is divided into 3 steps, and the 1st step is 95 DEG C of 5min, and the 2nd step is 95 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 2min, 35 circulations, and the 3rd step is 72 DEG C and extends 10min.Agarose gel electrophoresis reclaims PCR primer, reclaims product and connects pMD19-T cloning vector, and screening positive plasmid serves the order-checking of Hai Shenggong biotechnology company limited, and gained sequence is such as shown in SEQ ID NO.1.
The structure of embodiment 2. expression of recombinant e. coli carrier
By EcoR I and Sal I double digestion pcr amplification product and pET32a plasmid DNA respectively, agarose gel electrophoresis reclaims object band, connects the object band reclaimed, obtain prokaryotic expression carrier pET32a-bs1, use CaCl with T4DNA ligase enzyme
2method heat shock is transformed into intestinal bacteria BL 21 (DE3) for 90 seconds.Utilize Amp resistance screening list bacterium colony.Selected transformed clone through PCR, enzyme cut assay certificate clone correct after send Beijing Bo Maide biotechnology company limited to check order.Concrete structure flow process is shown in Fig. 1.
The conversion of embodiment 3. expression of recombinant e. coli carrier and screening
The correct recombinant expression vector pET32a-bs1 plasmid of qualification is proceeded to e. coli strain bl21, adds the IPTG abduction delivering 6h of 1mmol/L, add phosphoric acid buffer re-suspended cell.
The expression of embodiment 4. variant part S gene in intestinal bacteria
In the LB liquid nutrient medium of 10ml containing 100 μ g/ml Amp, inoculate BL21-pET32a-bs1 bacterium liquid by 1:1000,37 DEG C, 250rpm shakes overnight incubation; The bacterium liquid that next day spends the night by 1:50 inoculation culture extremely contains in the new LB liquid nutrient medium of 100 μ g/ml, 37 DEG C, and 250rpm concussion is cultured to OD
600=0.6 ~ 0.7; Often pipe adds IPTG respectively to final concentration is 1mmol/L, 37 DEG C of abduction deliverings; Bacterium liquid is collected, 4 DEG C, 8000rpm collected by centrifugation thalline respectively after induction 4h, 5h, 6h, 7h; Add phosphate buffered saline buffer (being called for short PBS, pH 7.4) the resuspended thalline of 10ml 0.01M.4 DEG C, the centrifugal 20min of 8000rpm, abandons supernatant.Repeated washing thalline once.Add the resuspended thalline of the above-mentioned damping fluid of 5ml.SDS-PAGE electrophoresis detection, as shown in Figure 2.Near 69kDa position, occur the specific band of a high expression level amount, molecular size range is about 69kDa, conforms to theoretical value.
Recombinant vectors pET32a-bs1 of the present invention can express acquisition recombinant protein, prepares protein immunization piglet in a small amount, within after exempting from 2 weeks, detects antibody, can produce specific antibody.Research for PEDV recombinant vaccine lays the foundation by achievement of the present invention.
SEQUENCE LISTING
<110> Xinjiang Tiankang Raise Livestock Biotechnology Co., Ltd
The construction process of a <120> grice diarrhoea virus variant part S prokaryotic expression vector
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1476
<212> DNA
<213> grice diarrhoea virus variant part S gene
<400> 1
gaattcgatg tcaccaggtg ctcagctaac actaatttta ggcggttctt ttcaaaattt 60
aatgttcagg cgcctgcagt tgttgtactg ggcggttatt taccttttgg tgaaaaccag 120
ggtgtcaatt caacttggta ctgtgctggc caacatccaa ctgctagtgg cgttcatggt 180
atctttgtta gccatattag aggtggtcat ggctttgaga ttggcatttc gcaagagcct 240
tttgacccta gtggttacca gctttattta cataaggcta ctaacggtaa cactaatgct 300
actgcgcgac tgcgcatttg ccagtttcct agcattaaat cattgggccc cactgctaat 360
aatgatgtta caacaggtcg taattgccta tttaacaaag ccatcccagc tcatatgagt 420
gaacatagtg ttgtcggcat aacatgggat aatgatcgtg tcactgtctt ttctgacaag 480
atctattatt tttattttaa aaatgattgg tcccgtgttg cgacaaagtg ttacaacagt 540
ggaggttgtg ctatgcaata tgtttacgaa cccacctatt acatgcttaa tgttactagt 600
gctggtgagg atggtatttc ttatcaactc tgtacagcta attgcattgg ttattctgcc 660
aatgtatttg ctactgagcc caatggccac ataccagaag gttttagttt taataattgg 720
tttcttttgt ccaatgattc cactttggtg catggtaagg tggtttccaa ccaaccattg 780
ttggtcaatt gtcttttggc cattcctaag atttatggac taggccaatt tttctccttt 840
aatcaaacga tcgatggtgt ttgtaatgga gctgctgtgc agcgtgcacc agaggctctg 900
aggtttaata ttaatgacac ctctgtcatt cttgctgaag gctcaattgt acttcatact 960
gttttaggaa caaatttttc ttttgtttgc agtaattcct cagatcctca cttagccacc 1020
ttcgtcatac ctctgggtgc tatccaagta ccctattatt gttttcttaa agtggatact 1080
tacaactcca ctgtttataa attcttggct gttttacctc ctaccgtcag ggaaattgtc 1140
atcaccaagt atggtgatgt ttatgccaat gggtttggct acttgcatct cggtcttttg 1200
gatgctgtca caattaattt cactggtcat ggcactgacg atgacgtttc tggtttttgg 1260
accatagcat cgactaattt tgttgatgca cttatcgaag ttcaaggaac tgccattcag 1320
cgtattcttt attgtgatga tcctgttagc caactcaagt gttctcaggt tgcttttgac 1380
cttgacgacg gtttctaccc tatttcttct agaaaccttc tgagtcatga acagccaagt 1440
tcttttgtta ctttgccatc atttaattaa gtcgac 1476
Claims (3)
1. a grice diarrhoea virus variant part S gene, is characterized in that: described S gene is made up of the nucleotide sequence shown in SEQ ID NO.1 in sequence table.
2. a construction process for grice diarrhoea virus variant part S prokaryotic expression vector, is characterized in that: this construction process is made up of following steps:
(a) with F1 and R1 for primer, F1:5 '-GAATTCGATGTCACCAGGTGCT-3 '; R1:5 '-GTCGACTTAATTAAATGATGG-3 '; PCR reaction amplification is utilized to obtain the gene order of the coding grice diarrhoea virus variant part S gene as shown in SEQ ID NO.1; Described PCR reaction system is: 2 × Ex Taq PCR premix 10 μ l, cDNA 2 μ l, primer mixture 1 μ l, distilled water 7 μ l; Reaction parameter is divided into 3 steps, and the 1st step is 95 DEG C of 5min, and the 2nd step is 95 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 2min, 35 circulations, and the 3rd step is 72 DEG C and extends 10min;
B the grice diarrhoea variant part S gene order of above-mentioned coding is connected on pET32a carrier by (), obtain recombinant expression vector pET32a-bs1;
C recombinant expression vector pET32a-bs1 plasmid is proceeded to e. coli strain bl21 by (), 250rpm concussion is cultured to logarithmic phase OD
600=0.6 ~ 0.7, add 1mmol/L IPTG, 37 DEG C of induction 6h express the structure of target protein and grice diarrhoea virus variant part S prokaryotic expression vector, complete.
3. the expression method of a grice diarrhoea virus variant part S gene in intestinal bacteria, it is characterized in that: this expression method has the following steps composition: the e. coli bl21 bacterium colony described in (1) picking claim 2, access contains antibiotic LB substratum, overnight incubation; (2) getting overnight culture transfers containing in antibiotic fresh LB nutrient solution, and 250rpm concussion is cultured to logarithmic phase OD
600=0.6 ~ 0.7; (3) in culture, add the IPTG that concentration is 1mmol/L, 37 DEG C, after abduction delivering 6h, 8000rpm, 20min centrifugal treating collects the coli somatic precipitation containing restructuring grice diarrhoea virus variant part S gene.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106554944A (en) * | 2015-09-28 | 2017-04-05 | 普莱柯生物工程股份有限公司 | The vaccine combination and application of pig epidemic diarrhea virus attenuated strain and its preparation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088039A (en) * | 2013-01-14 | 2013-05-08 | 广东大华农动物保健品股份有限公司 | Amplification method of porcine epidemic diarrhea virus S-gene epitope |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088039A (en) * | 2013-01-14 | 2013-05-08 | 广东大华农动物保健品股份有限公司 | Amplification method of porcine epidemic diarrhea virus S-gene epitope |
Non-Patent Citations (2)
| Title |
|---|
| 毕玉敏等: "猪流行性腹泻病毒Ｓ蛋白部分抗原表位基因的原核表达及表达产物的免疫原性分析", 《中国兽医科学》 * |
| 王萃瑜: "猪流行性腹泻病毒部分S基因的原核表达及间接ELISA检测方法的建立", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106554944A (en) * | 2015-09-28 | 2017-04-05 | 普莱柯生物工程股份有限公司 | The vaccine combination and application of pig epidemic diarrhea virus attenuated strain and its preparation |
| CN106554944B (en) * | 2015-09-28 | 2020-04-14 | 普莱柯生物工程股份有限公司 | Porcine epidemic diarrhea virus low-virulent strain, vaccine composition prepared from porcine epidemic diarrhea virus low-virulent strain and application of vaccine composition |
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Application publication date: 20150218 |