CN1952156A

CN1952156A - Recombination and expression for non-antibiotic expression vector of rotavirus Vp6 gene and lactic acid bacteria

Info

Publication number: CN1952156A
Application number: CNA200510017199XA
Authority: CN
Inventors: 王春凤; 王会岩; 李玉
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2005-10-19
Filing date: 2005-10-19
Publication date: 2007-04-25

Abstract

The invention belongs to the field of biological gene, disclosing the recombination and expression of a rotavirus Vp6 gene and Bacterium acidi lactic nonresistance expression vector and it is characterized in: digesting the cloning vector and vector taking thyA gene as a selection pressure using Sac I and KpnI, purifying and recovering, linking the products with T4 DNA ligase, transforming to thyA gene-deficient E. coli competence E. coli X13, culturing in the medium, and screening to obtain prokaryotic recombinant expression plasmid through growing functional redeem, inducing positive bacteria for expression using threonine to obtain high expression of the recombinant plasmid. Molecular weight of fusion protein is about 44.88 KD. Beneficial effects are: VP6 protein is RV-group-specific antigen, locating at the virus endoconch, occupying 51%of the virus particles. As VP6 mainly stimulates organism to generate mucosal immune antibody sIgA, it plays a very important role in the mucosal immune, thus advanced development of rotavirus Vp6 mediated gene oral mucosal immune genetically engineered vaccine has important significance.

Description

The reorganization of rotavirus Vp6 gene and the non-resistance expression vector of milk-acid bacteria and expression

Technical field

The invention belongs to field of biological genes, be specifically related to gene order, the Vp6 gene of pig A group rotavirus Wa strain Vp6 gene the clone, and the non-antibiotic resistance of " vaccine level " can be between milk-acid bacteria and intestinal bacteria plasmid vector pW425t reorganization, lactobacillus, mucosa-immune, the expression in intestinal bacteria of shuttling expressing.

Background technology

(Rotavirus is to cause one of main pathogen that infant and various young animal non-bacterial are suffered from diarrhoea RV) to rotavirus.RV diarrhoea is a kind of Pandemic infection disease.From Mebus in 1969 etc. isolated rotavirus (RV) first from calf diarrhea since, people found that gradually this virus is to cause infant and young animal diarrhoea and the main pathogens that causes death and die.The infection that rotavirus causes causes nearly 1,000,000 infant's death every year in developing country.A group RV causes the cacatory most important cause of disease of whole world infant, in developing country, and annual generation moderate and about 18000000 people of cacatory children, 350000～390000 people that surpass that die from rotavirus infection; In developed country, be children's common disease of being in hospital because of, medical expense height because RV diarrhoea causes dewatering, the annual directly medicine of the U.S. is with a toll of 200,000,000 6 thousand ten thousand dollars according to estimates.In November, 2004, Guangzhou rotavirus day is infected nearly 1000 people.In February, 2005, Bangladesh has 190000 people and infects rotavirus, wherein 32 people's death.In animality diarrhoea, the normal infection morbidity of birth animal such as piglet, hoggerel, horse, dog, cat, rabbit, ox, chicken, squab turkey, Yokogawa monkey, mouse.After nineteen eighty-two, China was separated to the buffalo rotavirus first, find that in succession the RV of pig, ox, sheep, dog, rabbit and multiple bird infects report, and found or isolation identification virus.According to the investigation to rotavirus antibody in East China ox, the porcine blood serum, the antibody positive rate of milk cow, ox and swinery reaches 85.4%, 83.0%, 68.3% respectively.Show that China's animal rotavirus infection is more general, infection rate is also very high.Rotavirus is mainly encroached on children livestock and poultry in age, and the piglet rotavirus infection of 15 ~ 30 ages in days is the most general; The age of onset of ox does not wait to 60 ages in days from being born, but the highest with 15 ~ 40 age in days sickness rate; The vulnerable period of rabbit and chicken is 30 ~ 70 ages in days and 7 ~ 30 ages in days.Adult animals also has susceptibility to rotavirus.Piglet sharply descends because of diarrhoea makes immunity of organisms after infecting PRV, and easily the secondary various bacterial infects, and causes the piglet survival rate to descend survivor's poor growth or stagnation.In recent years, along with the increase of the quantity of raising pigs, the popular scope of this disease constantly enlarges, and sickness rate constantly increases.Its sickness rate reaches as high as 80%, 14 age in days can reach 100% with the case fatality rate of interior newborn piglet, and China is the big country of raising pigs, and the piglet survival rate is low to have caused serious economy loss to pig industry.It is generally acknowledged that RV only causes the Digestive tract infringement, but the RV infection that studies show that in recent years can cause many organ injuries, viremia is the approach that the RV multisystem is sent out.Behind the zoogenetic infection RV production and growth there are considerable influence, cause serious economy loss.The improvement of environmental health and water sanitary condition can not reduce the frequency of disease development of rotavirus.Owing to still there is not the specific medicament of treatment rotavirus at present, therefore prevention seems particularly important.For this reason, the scientist that repeatedly calls upon States of the World Health Organization develops effective Rotavirus Vaccine, and it is classified as one of vaccine project of override development.Point out that vaccine prevention is only the unique effective means of containment rotavirus diarrhea.Therefore think the generation of control rotavirus disease, must set about from the development of vaccine.Longitudinal research shows, no matter developed country or developing country, the superinfection of infant's rotavirus is all very common, especially in postnatal initial several years.Compare with primary infection, infection symptoms is generally gentle once more, perhaps is symptomless infection, and this result of study has been affirmed with the vaccine immunity baby can prevent severe rotavirus disease.According to estimates, if use effective vaccine, the annual life that can save 500000 ~ 1000000 children in the whole world.Vaccine prevention is this sick optimal selection of control.Though current attenuated vaccine plays an important role in the generation of this disease of prevention, it still exists the unstable relatively of attenuated vaccine, easily reversion, defectives such as latent infection.Along with molecular biological further investigation, the appearance of novel gene engineered vaccine provides one approach for the diagnosis and the prevention of porcine rotavirus.

Along with the progress of Protocols in Molecular Biology, RV main protection antigen gene is cloned and is checked order, and now clear and definite VP6 albumen is the RV group-specific antigen, is positioned at the inner casing of virus, accounts for 51% of virion.Because VP6 mainly stimulates body to produce mucosa-immune antibody sIgA, has important effect in mucosa-immune, thereby the oral recombinant vaccine of development rotavirus Vp6 gene mediated mucosa-immune has great importance.

But raising difficult questions for discussion of needing at present to solve in genetically engineered is purifying expressed proteins how; because the F-strain of used express recombinant protective antigen mainly is the attenuation pathogenic bacteria that lives in the genetically engineered, and the immune protective efficiency of bacterial vaccine often is directly proportional with the remaining virulence of bacterial strain.Be subjected to people's attention day by day so attempt to seek the F-strain that to express exogenous antigen and safety well.The important member of normal microflora in lactobacillus behaviour and the animal body; with the F-strain of lactobacillus as expression external source protective antigen gene; just the biological function of milk-acid bacteria and the specific immunity of external source function antigen gene can be combined; Christiaens (1992) etc. discovers: lactobacillus can be used as the genetic engineering bacterium expressed exogenous gene; with intestinal bacteria; the lactobacillus of comparing only has one deck cytolemma; target protein can direct secretion enter supernatant under the guiding of signal peptide, thereby obtains target protein easily.Allson researchs such as (1996) is pointed out; lactobacillus is the good conversion or the expression system of protective antigen gene or immune-regulating factor; with the lactobacillus is the endogenous or foreign protein of vector expression, has tempting prospect in fields such as functional food, health cares.Succeeding in developing of this class vaccine will be explored a new road for the development of new generation vaccine.

But mostly the humans and animals digestive tube symbiosis lactobacillus expression vector system of report is with the pressure of antibiotics resistance as the foreign gene stably express at present.Yet because constantly drift diffusion in environment of drug resistance gene may cause the destruction to the little ecology of environment, have serious consequences, thereby this class expression system also has very big distance from the requirement of " edibility ", can not be directly used in humans and animals.So making up with nutritional factor replaces antibiotic resistance gene to have very important significance as the selective pressure of vector expression.The non-antibiotic resistance is that the expression system of selective pressure is that house-keeping gene defective bacterial strain with bacterium is a recipient bacterium, is cloned into the complete recipient bacterium dcc gene of homology or allos on plasmid, as the pressure of foreign gene stably express.From Nakayama (1988) has been since selective pressure makes up the non-resistance expression system of Salmonella typhimurium with the Asd gene, and this type of expression system development is very fast.At present the system of comparative maturity comprises: and thymidylate synthase gene (thymidylatesynthase, thyA), Asd gene, beta-galactosidase gene, SSB gene, D-xylose isomerase gene, amber mutant, ochre mutant, nisin and other the factor gene etc. of nourishing and growing.

Summary of the invention

The objective of the invention is:

The invention provides the reorganization and the expression of a kind of rotavirus Vp6 gene and the non-resistance expression vector of milk-acid bacteria, promptly the gene order of pig A group rotavirus Wa strain Vp6 gene, and the non-antibiotic resistance of " vaccine level " can be between milk-acid bacteria and intestinal bacteria plasmid vector pW425t reorganization, lactobacillus, the expression in intestinal bacteria of shuttling expressing.

Technical scheme of the present invention is:

The reorganization of full gene of rotavirus Vp6 and the non-resistance expression vector of milk-acid bacteria is: use inverse transcription polymerase chain reaction RT-PCR technology amplification Vp6 gene from the RV that cultivates, open reading frame contains 1194bp, 397 amino acid of encoding, by the T-A clone technology, with the PCR product cloning to cloning vector pGEM-T carrier, obtain recombinant clone plasmid pGEM-T-Vp6, with SacI and KpnI double digestion pGEM-T-Vp6 with thymidylate synthase gene thyA be selective pressure the non-antibiotic resistance can be between milk-acid bacteria and intestinal bacteria the plasmid vector pW425t of shuttling expressing, after purifying reclaims, connect with the T4DNA ligase enzyme, connecting product is converted among the intestinal bacteria competence E.coli X13 of thyA gene defection type, go up cultivation at common LB solid medium (not adding thymus pyrimidine), remedy through the growth function, screening obtains protokaryon recombinant expression plasmid pW425t-Vp6.

The expression of rotavirus Vp6 gene and the non-resistance expression vector of milk-acid bacteria is:, show through the SDS-PAGE electrophoretic analysis that recombinant plasmid obtains to efficiently express with positive bacteria Threonine abduction delivering, the relative molecular weight of fusion rotein is about 44.88kD.

The inventor made up " vaccine level " with the thyA gene be selective marker can be on milk-acid bacteria and non-resistance expression vector of bacillus coli shuttle expression carrier pW425t and basis with Eimeria tenella S07 gene successful expression.Use inverse transcription polymerase chain reaction (RT-PCR) technology amplification porcine rotavirus Vp6 full-length gene.Using DNASTAR and DNAMAN software analyzes.The nucleotide sequence and the amino acid sequence corresponding of this Wa strain rotavirus Vp6 gene are:

1 atg?gag?gtt?ctg?tac?tct?cta?tca?aaa?act?ctc?aaa?gat?gct?aaa?gac

1 MET?Glu?Val?Leu?Tyr?Ser?Leu?Ser?Lys?Thr?Leu?Lys?Asp?Ala?Lys?Asp

49 aaa?att?gtc?gaa?ggc?aca?tta?tac?tcc?aat?gta?agt?gat?cta?att?caa

17 Lys?Ile?Val?Glu?Gly?Thr?Leu?Tyr?Ser?Asn?Val?Ser?Asp?Leu?Ile?Gln

97 caa?ttt?aat?caa?atg?ata?att?act?atg?aat?gga?aat?gag?ttc?caa?act

33 Gln?Phe?Asn?Gln?Met?Ile?Ile?Thr?Met?Asn?Gly?Asn?Glu?Phe?Gln?Thr

145 gga?gga?att?ggt?aat?cta?ccg?att?aaa?aat?tgg?aat?ttt?gat?ttt?gga

49 Gly?Gly?Ile?Gly?Asn?Leu?Pro?Ile?Lys?Asn?Trp?Asn?Phe?Asp?Phe?Gly

193 tta?ctt?gga?aca?act?cta?cta?aat?tta?gat?gct?aac?tac?gtc?gaa?acg

65 Leu?Leu?Gly?Thr?Thr?Leu?Leu?Asn?Leu?Asp?Ala?Asn?Tyr?Val?Glu?Thr

241 gcc?cgc?aat?aca?att?gat?tat?ttt?gta?gat?ttt?gta?gat?aat?gta?tgt

8l Ala?Arg?Asn?Thr?Ile?Asp?Tyr?Phe?Val?Asp?Phe?Val?Asp?Asn?Val?cys

289 atg?gac?gaa?atg?gtt?aga?gaa?tca?caa?aga?aat?gga?att?gca?cca?caa

97 Met?Asp?Glu?Met?Val?Arg?Glu?Ser?Gln?Arg?Asn?Gly?Ile?Ala?Pro?Gln

337 tca?gat?tca?ctt?ata?aag?tta?tca?ggc?att?aaa?ttt?aaa?aga?ata?aat

113 Ser?Asp?Ser?Leu?Ile?Lys?Leu?Ser?Gly?Ile?Lys?Phe?Lys?Arg?Ile?Asn

385 ttt?gac?aat?tca?tca?gaa?tac?ata?gag?aac?tgg?aat?ttg?caa?aat?aga

129 Phe?Asp?Asn?Ser?Ser?Glu?Tyr?Ile?Glu?Asn?Trp?Asn?Leu?Gln?Asn?Arg

433 aga?caa?aga?acg?ggt?ttt?aca?ttt?cat?aaa?cca?aac?att?ttc?cct?tat

145 Arg?Gln?Arg?Thr?Gly?Phe?Thr?Phe?His?Lys?Pro?Asn?Ile?Phe?Pro?Tyr

481 tea?gct?tca?ttc?acg?ttg?aac?aga?tca?caa?ccg?gct?cat?gat?aac?ttg

161 Ser?Ala?Ser?Phe?Thr?Leu?Asn?Arg?Ser?Gln?Pro?Ala?His?Asp?Asn?Leu

529 atg?ggt?acg?atg?tgg?ctc?aat?gcg?gga?tca?gaa?att?cag?gtc?gct?gga

177 Met?Gly?Thr?Met?Trp?Leu?Asn?Ala?Gly?Ser?Glu?Ile?Gln?Val?Ala?Gly

577 ttc?gac?tac?tca?tgt?gca?ata?aac?gcg?cca?gct?agt?acg?caa?caa?ttt

193 Phe?Asp?Tyr?Ser?Cys?Ala?Ile?Asn?Ala?Pro?Ala?Ser?Thr?Gln?Gln?Phe

625 gag?cat?att?gta?cag?ctt?cga?agg?gtg?ttg?act?aca?gct?aca?ata?act

209 Glu?His?Ile?Val?Gln?Leu?Arg?Arg?Val?Leu?Thr?Thr?Ala?Thr?Ile?Thr

673 ctt?tta?cca?gat?gca?gaa?aga?ttt?agt?ttt?cca?aga?gtg?att?aat?tca

225 Leu?Leu?Pro?Asp?Ala?Glu?Arg?Phe?Ser?Phe?Pro?Arg?Val?Ile?Asn?Ser

721 gct?gac?gga?gcg?act?aca?tgg?tac?ttc?aat?cca?gtg?att?ctt?aga?cca

241 Ala?Asp?Gly?Ala?Thr?Thr?Trp?Tyr?Phe?Asn?Pro?Val?Ile?Leu?Arg?Pro

769 aat?aac?gtt?gaa?ata?gag?ttt?cta?cta?aac?ggg?cag?ata?ata?aat?act

257 Asn?Asn?Val?Glu?Ile?Glu?Phe?Leu?Leu?Asn?Gly?Gln?Ile?Ile?Asn?Thr

817 tac?caa?gca?aga?ttt?gga?acg?atc?ata?gct?aga?aat?ttt?gat?aca?att

273 Tyr?Gln?Ala?Arg?Phe?Gly?Thr?Ile?Ile?Ala?Arg?Asn?Phe?Asp?Thr?Ile

865 aga?ttg?tca?ttt?cag?ttg?atg?aga?cca?cca?aat?atg?aca?cca?gct?gta

289 Arg?Leu?Ser?Phe?Gln?Leu?Met?Arg?Pro?Pro?Asn?Met?Thr?Pro?Ala?Val

913 gcg?gcg?tta?ttt?cca?aat?gcg?cag?cca?ttt?gaa?cat?cac?gca?aca?gta

305 Ala?Ala?Leu?Phe?Pro?Asn?Ala?Gln?Pro?Phe?Glu?His?His?Ala?Thr?Val

96l gga?ctc?acg?ctt?aga?att?gaa?tct?gca?gtt?tgt?gaa?tca?gta?ctt?gcc

32l Gly?Leu?Thr?Leu?Arg?Ile?Glu?Ser?Ala?Val?Cys?Glu?Ser?Val?Leu?Ala

1009 gac?gca?agc?gaa?aca?atg?cta?gca?aat?gtg?aca?tct?gtt?aga?caa?gaa

337 Asp?Ala?Ser?Glu?Thr?Met?Leu?Ala?Asn?Val?Thr?Ser?Val?Arg?Gln?Glu

1057 tac?gcg?ata?cca?gtt?gga?cca?gtt?ttt?cca?cca?ggt?atg?aat?tgg?act

353 Tyr?Ala?Ile?Pro?Val?Gly?Pro?Val?Phe?Pro?Pro?Gly?Met?Asn?Trp?Thr

1105 gat?ttg?atc?act?aac?tat?tcg?cca?tct?aga?gag?gat?aac?ttg?cag?cgt

369 Asp?Leu?Ile?Thr?Asn?Tyr?Ser?Pro?Ser?Arg?Glu?Asp?Asn?Leu?Gln?Arg

1153 gta?ttt?aca?gtg?get?tcc?att?aga?agc?atg?ttg?att?aag?tga

385 Val?Phe?Thr?Val?Ala?Ser?Ile?Arg?Ser?Met?Leu?Ile?Lys***

Dna sequence analysis shows that the open reading frame of this gene (Vp6) is 1194bp, and translation back amino acid length is 397.Compare with the aminoacid sequence of other RV strain Vp6 gene, the result shows that the amino acid of the pig Vp6 gene of testing strain and the amino acid identity of reporting the Vp6 gene both at home and abroad are 42.7-92.2%, wherein the highest with Maryland, US strain (NO.D00326) homology, be 92.2%; Be 91.2% with Britain's strain (NO.L29184) and Venezuela's strain (NO.AF317123) homology; With another Britain's strain (NO.L29186) homology be 90.7%; With the homology of Heilungkiang strain (NO.AY538664) be 90.5%; With Argentinian strain (NO.U10031) homology be 90.2%; Minimum with Brazilian strain (NO.M94157) and French strain (NO.M29287) homology, be respectively 42.8% and 42.7%.

The pW425t carrier is the plasmid of shuttling expressing between intestinal bacteria and milk-acid bacteria, so recombinant plasmid is at first at expression in escherichia coli, genetic background is clear, cultivation is convenient, easy and simple to handle because intestinal bacteria have, it is simple especially to use the calcium chloride method for transformation.Recombinant plasmid transformed is gone among the intestinal bacteria competence E.coli X13 of thyA gene defection type, remedy screening positive clone pW425t-Vp6 through the outgrowth function, whether can by milk-acid bacteria and colibacillary shuttle expression carrier pW425t, at first at expression in escherichia coli and whether have reactinogenicity by SDS-PAGE and Western-blot if detecting foreign gene Vp6.

Just mostly the humans and animals digestive tube symbiosis lactobacillus expression vector system of reporting at present is with the pressure of antibiotics resistance as the foreign gene stably express.Yet because constantly drift diffusion in environment of drug resistance gene may cause the destruction to the little ecology of environment, have serious consequences, thereby this class expression system also has very big distance from the requirement of " edibility ", can not be directly used in humans and animals.So making up with nutritional factor replaces antibiotic resistance gene to have very important significance as the selective pressure of vector expression.The non-antibiotic resistance is that the expression system of selective pressure is that house-keeping gene defective bacterial strain with bacterium is a recipient bacterium, is cloned into the complete recipient bacterium dcc gene of homology or allos on plasmid, as the pressure of foreign gene stably express.From Nakayama (1988) has been since selective pressure makes up the non-resistance expression system of Salmonella typhimurium with the Asd gene, and this type of expression system development is very fast.At present the system of comparative maturity comprises: and thymidylate synthase gene (thymidylatesynthase, thyA), Asd gene, beta-galactosidase gene, SSB gene, D-xylose isomerase gene, amber mutant, ochre mutant, nisin and other the factor gene etc. of nourishing and growing.

The mucosa-immune system of humans and animals is meant body and the surperficial immunity of the extraneous cavity mucous membrane (gastrointestinal tract mucosa, respiratory mucosa, reproductive tract mucous membrane etc.) that communicates.Be the first line of defence that the body opposing is infected, so the mucosa-immune system is playing an important role aspect the body opposing infection.

Porcine rotavirus Vp6 gene can be by recombinating with lactic acid bacteria expression vectors, in the digestive tube milk-acid bacteria, express, excretory VP6 albumen can be induced the sIgA antibody that produces anti-rotavirus by stimulating the relevant lymphoid tissue (GALT) of intestinal mucosa, has critical function in the opposing porcine rotavirus infects.

With cloned plasmids pGEM-T-Vp4 and with the thyA gene be the carrier pW425t of selective pressure all with SacI, KpnI double digestion, purifying connects with the T4 dna ligase after reclaiming.Connect product and be converted among the intestinal bacteria competence E.coli X13 of thyA gene defection type, go up at common LB solid medium (not adding thymus pyrimidine) and cultivate, remedy through the growth function, screening obtains protokaryon recombinant expression plasmid pW425t-Vp6.With positive bacteria Threonine abduction delivering.The SDS-PAGE electrophoretic analysis shows that recombinant plasmid obtains to efficiently express, and the relative molecular weight of fusion rotein is about 44.88kD.Western blot analyzes and shows that this expression product and porcine rotavirus polyclonal antibody react, and carrier proteins does not react with antibody, show that this fusion rotein has good antigenicity, provide foundation for next step pW425t-Vp6 expresses in the milk-acid bacteria F-strain, also lay the foundation for the oral rotavirus milk-acid bacteria vaccine of development.

The invention has the beneficial effects as follows:

1, intestines local mucous membrane immunologic mechanism

Local immunity mechanism plays an important role in mediate protection.That play a major role in the enteron aisle local immunity is IgA.VP6 is considered to the immunifacient most important albumen of RV, can secrete sIgA by stimulating mucosal, thereby the mediation mucosal immunity is resisted subinfection again.At intestinal tube, the generation of sIgA must be that the relevant lymphoid tissue (GALT) of antigenic stimulation intestines wall and the IgA of the dimer that induces produce cell and return and be fixed in the intestinal mucosa lamina propria.Some research thinks that the mucosal immunity of enteron aisle is likely the more persistent predictor of clinical immunity.The short-term protection that natural infection mediated is also relevant with sIgA in essence.Mucosal immunity is not only a humoral immunization, also comprises the effect of cellular immunization and other immune factors.

The sIgA antibody of mucous membrane or glandular secretion can be in conjunction with special epithelial cell (M cell), and is transported to epithelium through the M cell and arrives the mucous membrane Lymphoid tissue again.This transportation can make sIgA or IgA-antigenic compound and lymphocyte or antigen presenting cell reaction, exposes the IgA binding site ^[73]IgA binding site on B cell and the T cell (FcA acceptor) finds in different animals.After entering mucosal tissue, IgA antibody (link to each other with antigen or do not link to each other) can be brought into play multiple function.At first cause mucosal immunoreaction, secondly, because the IgA-antigenic compound can be transported to Lymphoid tissue under the abdomen through the M cell, the FcA acceptor of antigen presenting cell (B cell, scavenger cell) can be strengthened picked-up and handle antigen; Through this approach, the sIgA of re-uptake can form one and enlarge ring, enhancing immunity and secondary immunoresponse; At last, the IgA that absorbs once more of M cell can pass through the immune response of genotype network adjustment.Therefore, IgA can activate mucomembranous immune system by M cellular uptake approach.

1.1 the intestinal mucosa immunity is to the restitution of acute infection

Virus-specific sIgA and/or CTL are confirmed by the several discovery in the laboratory animal the importance that acute viral infection recovers.The mouse that produces virus-specific sIgA capability defect is developed into chronic toxin expelling behind infected mice RV.In addition, some reports support virus-specific CTL may play an important role in acute infection recovers, as, virus-specific CTL appears at mouse intestinal mucosa surface in infecting in the week; Can not produce the adult mice (β of RV specific CTL ₂-microglobulin gene disappearance mouse) toxin expelling prolongs several days; Can improve symptom of diarrhea for acute infection suckling mouse input virus-specific CTL.

1.2 the effect that mucosal immunity infects protection again

The morbidity that the RV natural infection can protect infection again to cause.The amount (being reflected in the ight soil) of the virus-specific sIgA that the intestinal mucosa surface exists can be predicted the protectiveness of morbidity, also can be predicted by the existence of virus-specific IgA in the serum.Because RV duplicates in the chorioepithelium cell of intestinal mucosa surface-ripe, generally can not be in blood flow or away from the position detection of enteron aisle to virus.Therefore, the protection of infecting is again necessarily mediated by the immune response that is active in the intestinal mucosa surface.In addition, in laboratory animal, the toxin expelling amount that virus-specific IgA can make infection again cause in intestinal mucosa surface and the serum reduces.Yet vaccine immunity test shows, has general between serum and enteron aisle RV specificity IgA antibody titers and the disease-resistant anti-toxin expelling protectiveness and nisi dependency.Vaccine immunity and natural infection result's difference may be different relevant to the reaction of homology and heterology virus infection with the host, and for example homology virus a lot (differs 10 than heterology virus by force in the epithelial adaptation fecundity of host's intestinal villus ⁴～10 ⁵); Also may be with research method different relevant; the for example asynchronism(-nization) of blood sample collection; the vaccine immunity blood specimen collection is in last clothes seedling after January; this may obey seedling with the first time and differ some months; and RV specificity IgA is short-lived in the serum, so at this moment the level of IgA can not accurately indicate disease-resistant provide protection.

(Rotavirus RV) belongs to Reoviridae (Reoviridae), rotavirus (Rotaviras) to rotavirus, worldwide is widely current as the main pathogen of infant and animal diarrhoea, and bring about great losses every year.Serious and do not have effective treatment means in view of its harm, the World Health Organization classifies the RV vaccine as one of vaccine project of override development, and especially the development of its recombinant vaccine is even more important.

Description of drawings

Fig. 1 is a pW425t-Vp6 plasmid construction collection of illustrative plates of the present invention

Embodiment

Embodiment 1:

1, target gene PCR amplification and clone

According to the gene order of (AF317123.) porcine rotavirus Wa strain Vp6 among the GenBank, design the PCR primer (synthetic) of a pair of this gene of amplification: P by Dalian TaKaRa company ₁: 5 '-GTG GAGCTCATGGAG GTT CTG TAC TCT TTA T-3 '; P ₂: 5 '-GTG GGTACCTCA CTT AAT CAACAT GCT TCT-3 ' (the line sequence is respectively SacI, KpnI restriction enzyme site).Press the Trizol kit method and extract the total RNA of porcine rotavirus; With total RNA is template, is primer with P1, P2, and the Vp6 gene is carried out the RT-PCR amplification.50 μ l reverse transcription reaction systems and reaction conditions are as follows: get 10 μ l mRNA, 75C heats 5min, makes the double-stranded RNA sex change, place cooled on ice rapidly after, add 5 * AMV Buffer10.0 μ l again; DNTPs (10mM each) 4.0 μ l; RNasin 1.0 μ l; Downstream primer 0.5 μ l; AMV (5Units/ μ l) 0.5 μ l; Olig (dT) 1.0 μ l; MgCl ₂(25mM) 4.0 μ l; Add DEPC water 19.0 μ l.42C reverse transcription 1h, 95C boil 5min deactivation AMV ThermoScript II.50 μ l PCR reaction systems and reaction conditions are as follows: the 0.5ml EP pipe that the DEPC that learns from else's experience handles adds 10 * PCR Buffer, 5.0 μ l in 50 μ l reaction systems; DNTPs (2.5mM) 3.0 μ l; Upstream primer 1.0 μ l; Downstream primer 1.0 μ l; CDNA 5.0 μ l; RNasin 1.0 μ l; MgCl ₂2.0 μ l; EX Taq Dnase 0.5 μ l; DEPC water 31.5 μ l.Reaction conditions is the pre-sex change 8min of 94C; 94C sex change 1.5min, the 55C 1.0min that anneals, 72C extends 2.0min, totally 35 circles; 72C extends 10min.After the PCR product is purified, be connected among the pGEM-T Vector, and be converted among the competence E.coliJM109 with the T4 dna ligase.Identify recombinant clone by plasmid extraction, restriction analysis and pcr amplification, obtain recombinant plasmid pGEM-T-Vp6, deliver to Dalian TaKaRa company and check order, and use DNASTAR software and analyze.

2, the expression of milk-acid bacteria and escherichia coli prokaryotic expression construction of recombinant plasmid and goal gene

To recombinant plasmid pGEM-T-Vp6 and with the thyA gene be the carrier pW425t of selective pressure all with SacI, KpnI double digestion, cut glue purification after, connect with the T4 dna ligase.Connect product and be converted among the intestinal bacteria competence E.coli X13 of thyA gene defection type, go up at common LB solid medium (not adding thymidine) and cultivate, remedy through the growth function, screening obtains protokaryon recombinant expression plasmid pW425t-Vp6.

The recombinant expressed bacterium of protokaryon is inoculated in the common LB liquid of 5ml (the not adding thymidine) substratum 37 ℃ of 200r/min overnight incubation.Get 2ml and be inoculated in the 100ml LB liquid nutrient medium, 37 ℃ of 250r/min are cultured to OD ₆₀₀Be 0.6～0.8, in substratum, add 40mM DL-Threonine.Get bacterium liquid one time every 1h, be taken to 10h.Induce the E.coli X13 10h that contains empty plasmid pW425t to compare with same method.

3, SDS-PAGE analyzes

Bacterium liquid OD with each temporal induction results ₆₀₀All transfer to 0.72, get bacterium liquid 1.2ml, 4 ℃ of centrifugal collection thalline.Add 100 μ l deionized water cracking bacteriums in the precipitation thalline, 2 * sds gel the sample-loading buffer that adds equivalent again behind the mixing, boils 5min in boiling water, the centrifugal 10min of 15000r/min gets supernatant 15 μ l and carries out 15%SDS-PAGE by method in " molecular cloning ".

4, Western blot analyzes

Expression product is behind SDS-PAGE, by method in " molecular cloning " with BIO-RAD system electrotransfer to the PVDF transfer film, after the bovine serum albumin sealing, the goat anti-rabbit igg that adds the anti-pig polyclonal antibody of rotavirus rabbit, horseradish peroxidase-labeled successively, colour developing and observations in p-diaminodiphenyl (DAB) solution at last.

5, result

5.1 the RNA of rotavirus extracts

11 sections were the 4-2-3-2 distribution pattern by the molecular weight size when this experiment strain met A group RV nucleic acid electrophoresis.They are 6 coding structure albumen VP1-VP4, VP6, VP7 wherein, 5 codings Nonstructural Protein NSP1 (NS53), NSP2 (NS35), NSP3 (NS34), NSP4 (NS28) and NSP5 (NS26), wherein 4 kinds of albumen VP6, VP4, VP7 and NSP4 play an important role.

5.2RT-PCR amplification

, checking in 1% sepharose through pcr amplification for after the template reverse transcription becomes cDNA with the total RNA that extracts, is Marker with DL2000, and the result amplifies and the band of expecting that size conforms to.

5.3 the segmental recovery purifying of purpose

Reclaim test kit with DNA the purpose fragment is carried out purifying, the product behind the purifying is got 1 μ l and is checked in 1% sepharose, and result's specific band nothing but exists.

5.4 the evaluation of recombinant plasmid

5.4.1 the extraction of recombinant plasmid is identified

Picking is 7 white colonies and 1 blue colonies wherein, inoculates in the common AMP+LB nutrient solution, after 37 ℃ of incubated overnight, carries out the little extracting of plasmid.Through 1% agarose gel electrophoresis, the result shows that the plasmid of 1,3,4,5 bacterium colonies may contain the purpose fragment.

5.4.2 enzyme is cut evaluation

Select that wherein 1, No. 3 bacterium carries out SacI and the KpnI enzyme is cut evaluation.The result shows, contains foreign gene Vp6 in the recombinant vectors in the bacterial strain 3, positive clone; No external source Vp6 gene in the bacterial strain 1, negative clone.

5.4.3 PCR identifies

Plasmid with No. 3 bacterium is a template, carries out PCR and identifies, expands the purpose band that conforms to the expection size.

5.5 the structure of milk-acid bacteria and intestinal bacteria protokaryon recombinant expression plasmid and the evaluation of recon

PGEM-T-Vp6 and pW425t are all with behind SacI, KpnI double digestion and the purifying, connect by the T4 dna ligase, connecting product is converted among the intestinal bacteria competence E.coli X13 of thyA gene defection type, the E.coli X13 of thyA genetic flaw can not grow or poor growth on common LB substratum, must could recover growth under the condition that is added with the thymidine of external source (50 μ g/ml).Contain the E.coli X13 that changing over to of thyA gene recombination plasmid pW425t-Vp6 can make the thyA genetic flaw in this experiment and on common LB flat board, recover growth.

To (not add in the thymidine (50 μ g/ml) of external source and cultivate at common LB liquid nutrient medium through bacterium colony that the outgrowth function remedies acquisition, 7 bacterium colonies to screening carry out the plasmid extraction, bacterial strain 1,2 may contain the plasmid of expection, further identifies but still need.With SacI and KpnI double digestion, obtain the insertion fragment of pW425t linear fragment and the 1194bp of about 3700bp, show the milk-acid bacteria and the intestinal bacteria protokaryon recombinant expression plasmid pW425t-Vp6 that construct porcine rotavirus Vp6 gene.

5.5.1 the PCR of recombinant expression plasmid pW425t-Vp6 identifies

Recombinant plasmid 1 with primary dcreening operation is a template, pcr amplification Vp6 gene, and the result amplifies the purpose band and specific band nothing but.

5.5.2 the enzyme of recombinant expression plasmid pW425t-Vp6 is cut evaluation

Positive recombinant plasmid 1 usefulness SacI and KpnI enzyme are cut deserved two bar segment: big fragment is about 3700kb, and small segment is about 1194bp, and enzyme is cut the result and conformed to expection.

5.6 SDS-PAGE analyzes

Analyze the expression of above-mentioned process each time that is accredited as male clone strain 1 in E.coli X13.The Vp6 gene has obtained expression, compared with the control, its expression amount in time prolongation and increase, proteinic relative molecular weight is about the 44.88kD fusion rotein, and is consistent with expection VP6 proteic molecular weight.This protein expression is not then seen in the E.coli X13 contrast that contains empty plasmid pW425t.

5.7 Western blot analyzes

Expression product is behind the SDS-PAGE electrophoresis, be transferred on the PVDF transfer film, with the anti-porcine rotavirus polyclonal antibody of rabbit is one anti-, the goat anti-rabbit igg of horseradish peroxidase-labeled is two anti-, p-diaminodiphenyl (DAB) is substrate, carry out western blot analysis, as seen the result has a tangible Western blot band at the 44.88kD place, illustrates that this expression product and porcine rotavirus polyclonal antibody have reactionogenicity.

Claims

1, the reorganization and the expression of a kind of rotavirus Vp6 gene and the non-resistance expression vector of milk-acid bacteria is characterized in that:

The reorganization of rotavirus Vp6 gene and the non-resistance expression vector of milk-acid bacteria is: use inverse transcription polymerase chain reaction RT-PCR technology amplification Vp6 gene from the RV that cultivates, open reading frame contains 1194bp, 397 amino acid of encoding, by the T-A clone technology, with the PCR product cloning to cloning vector pGEM-T carrier, obtain recombinant clone plasmid pGEM-T-Vp6, with SacI and KpnI double digestion pGEM-T-Vp6 with thymidylate synthase gene thyA be selective pressure the non-antibiotic resistance can be between milk-acid bacteria and intestinal bacteria the plasmid vector pW425t of shuttling expressing, after purifying reclaims, connect with the T4DNA ligase enzyme, connecting product is converted among the intestinal bacteria competence E.coli X13 of thyA gene defection type, go up cultivation at common LB solid medium (not adding thymus pyrimidine), remedy through the growth function, screening obtains protokaryon recombinant expression plasmid pW425t-Vp6;

2, the reorganization and the expression of a kind of rotavirus Vp6 gene according to claim 1 and the non-resistance expression vector of milk-acid bacteria is characterized in that:

From the RV that cultivates, use inverse transcription polymerase chain reaction RT-PCR technology amplification Vp6 gene, open reading frame contains 1194bp, 397 amino acid of encoding, by the T-A clone technology, with the PCR product cloning to cloning vector pGEM-T carrier, obtain recombinant clone plasmid pGEM-T-Vp6, use DNASTAR and DNAMAN software and analyze, the nucleotide sequence and the amino acid sequence corresponding of this Wa strain rotavirus Vp6 gene are: