CN102382812A - Reverse genetics operating system of infectious bronchitis virus H120 vaccine strain and operating method thereof - Google Patents
Reverse genetics operating system of infectious bronchitis virus H120 vaccine strain and operating method thereof Download PDFInfo
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Abstract
The invention relates to a reverse genetics operating system of infectious bronchitis virus H120 vaccine strains and an operating method thereof; the system comprises a plurality of recombinant plasmids which are used for constructing a genome full-length cDNA of the infectious bronchitis virus H120 vaccine strains, and assistant cDNAs of the recombinant plasmids, wherein the assistant cDNAs encode nucleoproteins (N) of the infectious bronchitis virus H120 vaccine strains. H120 virus strains which acquire gene mutation or gene replacement can be successfully rescued by adopting the reverse genetics operating system. The reverse genetics operating system of the infectious bronchitis virus H120 vaccine strains and the operating method thereof provide a firm foundation for researching molecular virology of the infectious bronchitis virus. The invention also relates to an application for disease diagnosis and vaccine development generated and derived by the reverse genetics operating system.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of IBV H120 vaccine strain reverse genetic operating system and working method thereof.
Background technology
(Infectious Bronchitis IB) is avian infectious bronchitis virus (Infectious Bronchitis Virus, a kind of acute height contagious disease that IBV) causes by the coronaviridae coronavirus genus to infectious bronchitis.This virus can infect the chicken of all ages in days, causes growth retardation, death and the price of deed to reduce; Also, bring massive losses to aviculture usually because of secondary infections such as Mycoplasma polyinfection and colibacillosis improve chicken crowd's mortality ratio.At present, this disease is the world and is widely current, and is one of serious infectious diseases of serious harm world aviculture.
The genome of IBV is the sub-thread positive chain RNA, is about 27.6kb, has typical 5 ' cap and 3 ' Poly A tail structure, has 10 tangible ORFs (ORF) at least, respectively the structural protein and the Nonstructural Protein of coding virus.Wherein S albumen has constituted the fine shape projection in top layer of IBV; Be most important protective antigen, its effect shows as stimulates body to produce neutralizing antibody, in the virus absorption onto cell process, plays a role; The tissue tropism of therefore decision virus, but be not unique deciding factor of IBV virulence.N albumen is the constitutive protein of IBV virus inner core capsid, in virus replication, assembling, effect is arranged, and proteic phosphorylation has vital role to the performance of its function.3a, 3b, 5a and 5b are the Nonstructural Protein of encoding viral, in the IBV cells infected, all can detect, but its function are not clear at present.The disappearance of recent findings 3a, 3b, 5a and 5b coding region does not influence the pathogenic of duplicating of IBV virus and virus.
IBV rna gene group is transcribed mechanism because of having unique guide's primer, has very high mispairing rate.Under the influence of factors such as immunization program, poultry density, IBV just morphs with different speed and different Evolution direction.Isolating in the world at present main epidemic strain has M41 strain, Conn strain, Australian T strain, Hotle strain, Gray strain etc., and different strains exist very big-difference in virulence, pathogenic and tissue tropism.Vaccine inoculation is the important means of prevention IB, develops conventional IB seedling alive and deactivation vaccine in succession, and malicious seedling was by the countries in the world widespread use a little less than wherein the chicken embryo adapted to malicious H120 and H52.Because a little less than the cross protection power, this has brought very big difficulty for the immunoprophylaxis of IB between the different serotypes IBV strain.Conventional single strain vaccine often has the report of immuning failure, and less toxic vaccine also exists reorganization, virulence to return problems such as strong.It is extremely urgent to develop effective novel vaccine to the new popularity of IBV.
Reverse genetic manipulation technology (reverse genetics); Promptly the full-length infectious CDNA clones of virus is technological; Comprise viral genome full length cDNA clone constructing technology and transcribe the infectious transcript technology of preparing of viral RNA by cDNA; Can on the viral DNA level, carry out manual operation, solve the unworkable difficult problem of rna virus cdna group, thereby strong means are provided for development of the gene replication of RNA viruses and the research of expression regulation mechanism, novel vaccine etc. it.The genome of IB virus is a viroid maximum in the present known RNA viruses, and its reverse genetic manipulation technical difficulty is bigger.The reverse genetic operating system of IBV H120 vaccine strain has been set up in this research, for solid basis has been established in the development of this disease new generation vaccine.
Summary of the invention
An object of the present invention is to provide a kind of IBV H120 vaccine strain reverse genetic operating system, this system comprises:
1) the full-length cDNA molecule of structure RNA viruses, its 5 ' end of this full-length cDNA molecule has the core sequence of complete T7 rna polymerase promoter, and 3 ' end has polyA tail structure;
2) the encode cDNA sequence of nucleoprotein (N) of said IBV H120 vaccine strain of auxiliary cDNA, auxiliary cDNA, its 5 ' end has the core sequence of complete T7 rna polymerase promoter, and 3 ' end has polyA tail structure.
3) transfectional cell.
Base genomic the 738th in the full-length cDNA molecule sports T from C.
Base genomic the 17243rd in the full-length cDNA molecule sports T from G.
CDNA sequence two ends in a plurality of recombinant plasmids of structure full-length cDNA molecule have the restriction enzyme enzyme recognition site, to realize the intersegmental directed cloning of sheet, are used to make up the full-length cDNA molecule of said IBV H120 vaccine strain.
Described restriction enzyme is Bsa I.
In the full-length cDNA molecule 5a gene is replaced to the green fluorescence gene.
Described IBV is the H120 vaccine strain.
Wherein said transfectional cell is BHK-21.
A kind of method of reverse genetic operating system rescue IBV is characterized in that, carries out according to the following steps:
1) after being cut, said a plurality of recombinant plasmid enzymes connect into the full-length cDNA molecule;
2) with full-length cDNA and said auxiliary cDNA after in-vitro transcription becomes RNA, the host cell of cotransfection virus replication permission;
3) results host cell and supernatant, the inoculated into chick embryo allantoic cavity is rescued and is obtained virus.
Said 1) be that the template reverse transcription becomes cDNA with the H120 strain virus geneome RNA that extracts earlier in the step; Then said cDNA is carried out pcr amplification, the base mutation of 17243 of viral genome in the amplification procedure; Be connected to the laggard row filter of pMD19-T carrier after the amplification, carry out the base mutation of 738 of viral genome after the screening; Then inoculate the LB substratum and cultivate, reclaim the purpose fragment according to method then; Last each purpose fragment connects into the full-length cDNA molecule through restriction enzyme site.
Said 2) the H120 strain virus geneome RNA with extraction is that the template reverse transcription becomes cDNA in the step, carries out N protein gene pcr amplification then, then carries out Hind III and Xho I double digestion; The pVAX1 carrier carries out Hind III and Xho I double digestion equally simultaneously; Then both are connected and obtain recombinant plasmid pVAXN; PVAXN with screening is a template at last, reclaims the auxiliary cDNA that contains the N gene behind the pcr amplification.
Make up in the full-length cDNA molecular process 5a gene is replaced to the green fluorescence gene.
1) in the step H120 pnca gene group being divided into 10 sections according to the distribution situation of IIs type restriction enzyme Bsa I increases; Then will according to the method in the claim 10 obtain purpose fragment TM1, TM2 ..., TM10; According to volumetric molar concentration 1: 1 TM2 is mixed with TM3, TM4 and TM5, TM6 and TM7, TM8 and TM9 at last and be connected; Obtain intermediate product TM2-3, TM4-5, TM6-7, TM8-9; Then with TM1, TM2-3, TM4-5; TM6-7, TM8-9, TM10 connect according to 1: 1: 1 mixed of volumetric molar concentration respectively, obtain intermediate product TM1-5, TM6-10, TM1-5, TM6-10 are connected according to 1: 1 mixed of volumetric molar concentration to obtain the full-length cDNA molecule at last.
Description of drawings
The strategy of the full genome segmentation amplification of Fig. 1 H120 virus strain.According to the distribution situation of IIs type restriction enzyme Bsa I, divide 10 fragments such as TM1a, TM2, TM3, TM4, TM5, TM6, TM7, TM8, TM9 and TM10 that full genome is carried out the segmentation amplification.
The full genome RT-PCR of Fig. 2 H120 virus strain segmentation amplification.RT-PCR obtains 10 dna fragmentation TM1a, TM2, TM3, TM4, TM5, TM6, TM7, TM8, TM9 and TM10; Segmental size is consistent with theoretical value, is respectively 0.9kb, 3.7kb, 2.9kb, 2.6kb, 3.7kb, 3.7kb, 3.6kb, 3.2kb, 3.1kb, 0.9kb.Wherein M is DL5000 Marker.
The strategy of the full genome cDNA segmentation splicing of Fig. 3 H120 virus strain.Wherein base represented in the vertical letter of picture, is the intersegmental terminal complementary sequence that is used for connecting of sheet; According to the volumetric molar concentration of dna fragmentation, according to 1: 1 ratio TM2 and TM3, TM4 and TM5, TM6 and TM7, TM8 and TM9 are mixed, utilize the intersegmental restriction enzyme site of each sheet to connect, obtain intermediate product TM2-3, TM4-5, TM6-7, TM8-9; Once more with TM1, TM2-3 and TM4-5, TM6-7, TM8-9 and TM10 are connected after according to 1: 1: 1 mixed, obtain intermediate product TM1-5, TM6-10; At last TM1-5, TM6-10 are connected after according to 1: 1 mixed, obtain IBV H120 pnca gene group full-length cDNA, its 5 ' end has the core sequence of complete T7 rna polymerase promoter, and 3 ' end has polyA tail structure.
Fig. 4 obtains the synoptic diagram that the 5a gene replaces to the full genome cDNA of H120 strain of green fluorescence gene.Adopt high-fidelity pcr amplification green fluorescence gene (EGFP); With the 5a gene in its replacement pMDTM9 plasmid; Obtain the IBV H120 pnca gene group full-length cDNA that the 5a gene replaces to the green fluorescence gene through external splicing again; Its 5 ' end has the core sequence of complete T7 rna polymerase promoter, and 3 ' end has polyA tail structure.
Fig. 5 saves the order-checking qualification result of viral H120-R.Sequencing result shows that the base of the 738th of H120-R pnca gene group is T, but not the C of its parent H120 strain, conforms to the genetic marker result who sets, and the Bsa I restriction enzyme enzyme recognition site at this place has been eliminated in sudden change; Sequencing result shows that the base of the 17243rd of H120-R pnca gene group is T, but not the G of its parent H120 strain, conforms to the genetic marker result who sets, and a Bsa I restriction enzyme enzyme recognition site has been introduced in the sudden change at this place.
Fig. 6 saves the fluoroscopic examination result of viral H120-5a/EGFP infected chicken embryo.Get the tissues such as throat mucus, chorioallantoic membrane, amnion and mesentery of H120-5a/EGFP kind poison inoculated into chick embryo; Under the fluorescence inverted microscope after blue-light excited; All can in a large amount of cells, detect green fluorescence, show the H120-5a/EGFP recombinant virus can be in infected chicken embryo expressing green fluorescent protein.
What Fig. 7 saved viral H120-R, H120-5a/EGFP causes the test of short and smallization of chicken embryo.The result all can cause chicken embryo death after showing H120-R and H120-5a/EGFP infected chicken embryo, and characteristic change occurs: amnion thickens, and is close to idiosome; Chick embryo development is obstructed; Idiosome is curled than short and small with the normal chicken embryo of age in days, with its parent's strain H120 pathogenic identical to the chicken embryo.
Fig. 8 saves the mensuration of viral H120-R, H120-5a/EGFP growth curve in the chicken embryo.The virus titer of different time sections behind employing fluorescence quantitative RT-RCR mensuration H120-R, H120-5a/EGFP and the parent's strain H120 infected chicken embryo thereof; The result shows that the virus titer of H120-R and parent's strain H120 thereof reaches the climax behind inoculated into chick embryo 36h, descends gradually then; And the virus titer of H120-5a/EGFP reaches the climax behind inoculated into chick embryo 48h.
Embodiment
Following examples are described the present invention in detail, and said embodiment only is meant for illustration the present invention, but is not used for limiting scope of the present invention.Scope of the present invention is specifically limited accompanying Claim.
1 makes up the full-length gene group cDNA of IBV H120 strain
The genome size of IB virus is about 27.6kb, and different isolates is slightly variant on length.According to the distribution situation of the last IIs type restriction enzyme BsaI of IBV H120 vaccine strain genom sequence (GenBank accession number FJ807652), divide 10 fragments such as TM1a, TM2, TM3, TM4, TM5, TM6, TM7, TM8, TM9 and TM10 that full genome is carried out segmentation amplification (Fig. 1).Design covers should the complete genomic 10 pairs of primers of virus; Primer sequence and the position in genome (seeing table 1) thereof: in the upstream primer TM1S of TM1a amplified fragments, introduce the core sequence of T7 rna polymerase promoter, after guaranteeing the accomplishing full-length cDNA splicing, can produce virus genome RNA in in-vitro transcription; Primer MU1S, MU1A sport T with the base C of the 738th of H120 strain virus genome; Eliminated the Bsa I restriction enzyme enzyme recognition site (GGTCTC → GGTCTT) at this place; Codon CTC and the sudden change back codon CTT Leu that all encodes before the sudden change; Be same sense mutation, can be as the genetic marker of rescue virus, with the dna fragmentation called after TM1 after the sudden change; In primer TM6A, TM7S, introduce point mutation; Bases G with the 17243rd of H120 strain virus genome after the amplification sports T; Codon GGG and the sudden change back codon GGT Gly that all encodes is same sense mutation before the sudden change, and the sequence GGTCTC of acquisition is a Bsa I restriction endonuclease recognition sequence; Can be used in the structure of full-length cDNA, also can be as the genetic marker of rescue virus; 5 ' end at primer TM1S, TM4A, TM5S, TM5A, TM6S, TM8A, TM9S and TM10A is introduced Bsa I restriction endonuclease recognition sequence respectively, to guarantee the splicing of follow-up full-length cDNA; In downstream primer TM10A, introduce 25 base T, to guarantee having polyA tail structure at TM10 fragment 3 ' end.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Table 1 H120 genome full-length cDNA amplimer
Annotate: tilted letter is the T7 rna polymerase promoter sequence; Underlined letter is the restriction enzyme enzyme recognition sequence.
With IBV H120 vaccine strain inoculated into chick embryo allantoic fluid, adopt RNAisoPlus (TaKaRa) to extract geneome RNA according to the step in the test kit working instructions.The RNA that obtains dissolves with 20 μ L RNase-free water, in-70 ℃ of preservations.
H120 strain virus geneome RNA to extract is a template; PrimeScriptlst Strand cDNA Synthesis test kit (TaKaRa) is adopted in reverse transcription, makes up the reaction system of 20 μ L according to working instructions, with Oligo dT and Random Primers as the reverse transcription primer; Carry out 30 ℃ earlier; The 10min reaction, 42 ℃ then, the 60min reverse transcription reaction.After reaction finished, 70 ℃, the 15min inactivation was handled, in-20 ℃ of preservations.
With above-mentioned cDNA is template, and the primer in the employing table 1 divides 10 fragments to TM1S+TM1A, TM2S+TM2A, TM3S+TM3A, TM4S+TM4A, TM5S+TM5A, TM6S+TM6A, TM7S+TM7A, TM8S+TM8A, TM9S+TM9A and TM10S+TM10A, and (TM1a～TM10) carries out pcr amplification.High-fidelity enzyme PrimeSTAR HS DNA Polymerase (TaKaRa) is adopted in the PCR reaction, makes up the reaction system of 50 μ L according to product description.Reaction conditions is: 95 ℃, and 25s; 52 ℃～58 ℃, 25s; 72 ℃, 1min～3min.The PCR product carries out glue and reclaims after 1% agarose gel electrophoresis detects; Obtain 10 dna fragmentation TM1a, TM2, TM3, TM4, TM5, TM6, TM7, TM8, TM9 and TM10; Segmental size is consistent with theoretical value, is respectively 0.9kb, 3.7kb, 2.9kb, 2.6kb, 3.7kb, 3.7kb, 3.6kb, 3.2kb, 3.1kb, 0.9kb (Fig. 2).
After above-mentioned 10 dna fragmentations are added T and handle with the Taq enzyme, be connected respectively in the pMD19-T carrier (TaKaRa), transform the JM109 genetic engineering bacterium, enzyme is cut the evaluation positive colony.The positive colony that obtains is served sea living worker's biotechnology Services Co., Ltd carry out sequencing, in full accord to confirm cDNA fragment and the virus genome sequence inserted among recombinant plasmid pMDTM1a, pMDTM2, pMDTM3, pMDTM4, pMDTM5, pMDTM6, pMDTM7, pMDTM8, pMDTM9 and the pMDTM10.The TM1a dna fragmentation in the pMDTM1a recombinant plasmid wherein, its 5 ' end has the core sequence of complete T7 rna polymerase promoter; TM10 dna fragmentation in the pMDTM10 recombinant plasmid, its 3 ' end has polyA tail structure.
Recombinant plasmid pMDTM1a with screening is a template; Working instructions according to TaKaRa MutanBEST test kit (TaKaRa); Adopt primer MU1S, MU1A that the C of the 738th of H120 strain virus genome is sported T, eliminate Bsa I restriction enzyme enzyme recognition site (GGTCTC → GGTCTT), codon CTC and the sudden change back codon CTT Leu that all encodes before the sudden change at this place; Be same sense mutation, can be as the genetic marker of rescue virus.Transform the JM109 genetic engineering bacterium, enzyme is cut the evaluation positive colony.The positive colony that obtains is served sea living worker's biotechnology Services Co., Ltd carry out sequencing; Called after pMDTM1; Only there are well-determined mutational site in cDNA fragment and virus genome sequence to insert among definite recombinant plasmid pMDTM1, and its 5 ' end has the core sequence of complete T7 rna polymerase promoter.
The JM109 genetic engineering bacterium that carries recombinant plasmid is inoculated the LB substratum respectively cultivate, adopt TaKaRa MiniBEST Plasmid Purification Kit (TaKaRa) to extract recombinant plasmid pMDTM1, pMDTM2, pMDTM3, pMDTM4, pMDTM5, pMDTM6, pMDTM7, pMDTM8, pMDTM9 and pMDTM10 according to working instructions.Respectively to above-mentioned recombinant plasmid after Bsa I enzyme is cut, adopt Agarose Gel DNA Fragment Recovery Kit (TaKaRa) to reclaim purpose fragment TM1, TM2, TM3, TM4, TM5, TM6, TM7, TM8, TM9 and TM10 according to working instructions glue.
Adopt ultraviolet spectrophotometer to measure the mass concentration of above-mentioned 10 dna fragmentations, and be converted into volumetric molar concentration.Volumetric molar concentration according to dna fragmentation; According to 1: 1 ratio TM2 and TM3, TM4 and TM5, TM6 and TM7, TM8 and TM9 are mixed; Utilize the intersegmental restriction enzyme site of each sheet to connect, obtain intermediate product TM2-3, TM4-5, TM6-7, TM8-9 and carry out glue recovery and concentration determination.Once more with TM1, TM2-3 and TM4-5, TM6-7, TM8-9 and TM10 are connected after according to 1: 1: 1 mixed, obtain intermediate product TM1-5, TM6-10 and carry out glue and reclaim and concentration determination.At last TM1-5, TM6-10 are connected after according to 1: 1 mixed, obtain IBV H120 strain full-length cDNA (Fig. 3).
2 make up the H120 strain full-length gene group cDNA that the 5a gene replaces to the green fluorescence gene
The 5a gene is the nonstructural protein gene of IBV, and existing research shows and can lack and do not influence the propagation of virus on cell.According to the whole genome sequence (GenBank accession number FJ807652) of IBV H120 vaccine strain and the sequence of green fluorescence expression vector pEGFP-N1 (Clontech), be designed for and make up the primer (seeing table 2) that the 5a gene replaces to green fluorescence gene recombination plasmid pMDTM9-5a/EGFP.The 5a gene is arranged in the recombinant plasmid pMDTM9 of 1 structure, and 1 primer 5aA and 5aS are respectively designed in trip and downstream above that.Green fluorescence gene (EGFP) is arranged in the pEGFP-N1 carrier, and 1 primer EGFPS and EGFPA are respectively designed in trip and downstream above that.All primer 5 ' ends are all introduced Bsa I restriction endonuclease recognition sequence, to guarantee the splicing of follow-up TM9-5a/EGFP mosaic gene.
Table 2 construction recombination plasmid pMDTM9-5a/EGFP amplification primers
Annotate: underlined letter is the restriction enzyme enzyme recognition sequence; "
*" represent that primer sequence is arranged in the position of pEGFP-N1 carrier; "
*" represent that primer sequence is arranged in the position of H120 strain virus genome.
Be template with recombinant plasmid pMDTM9 and pEGFP-N1 carrier respectively, the primer in the employing table 2 carries out pcr amplification.High-fidelity enzyme PrimeSTAR HS DNAPolymerase (TaKaRa) is adopted in the PCR reaction, makes up the reaction system of 50 μ L according to product description.Reaction conditions is: 95 ℃, and 25s; 52 ℃, 25s; 72 ℃, 1min～3min.The PCR product of pMDTM9 and pEGFP-N1 adopts Bsa I restriction enzyme to carry out enzyme and cuts after 1% agarose gel electrophoresis detects.Above-mentioned two kinds of enzymes are cut product glue reclaim the back mixing, carry out ligation, transform the JM109 genetic engineering bacterium then, PCR identifies positive colony.With the recombinant plasmid called after pMDTM9-5a/EGFP that obtains, serve sea living worker's biotechnology Services Co., Ltd and carry out sequencing.
The JM109 genetic engineering bacterium inoculation LB substratum that carries the pMDTM9-5a/EGFP recombinant plasmid is cultivated, adopted TaKaRa MiniBEST Plasmid Purification Kit (TaKaRa) to extract recombinant plasmid pMDTM9-5a/EGFP according to the test kit working instructions.After the BsaI enzyme is cut, adopt Agarose Gel DNA Fragment Recovery Kit (TaKaRa) to reclaim target DNA fragment TM9-5a/EGFP to recombinant plasmid according to working instructions glue.
Ultraviolet spectrophotometer is measured the mass concentration of TM9-5a/EGFP dna fragmentation, and is converted into volumetric molar concentration.TM1, TM2, TM3, TM4, TM5, TM6, TM7, TM8 and TM10DNA fragment that employing 1 is obtained; According to segmental volumetric molar concentration; According to 1: 1 ratio TM2 and TM3, TM4 and TM5, TM6 and TM7, TM8 and TM9-5a/EGFP are mixed; Utilize the intersegmental restriction enzyme site of each sheet to connect, obtain intermediate product TM2-3, TM4-5, TM6-7, TM8-9/EGFP and carry out glue recovery and concentration determination.Once more with TM1, TM2-3 and TM4-5, TM6-7, TM8-9/EGFP and TM10 are connected after according to 1: 1: 1 mixed, obtain intermediate product TM1-5, TM6-10/EGFP and carry out glue and reclaim and concentration determination.At last TM1-5, TM6-10/EGFP are connected after according to 1: 1 mixed, obtain the IBV H120 strain full-length gene group cDNA (Fig. 4) that the 5a gene replaces to the EGFP gene.
The nucleoprotein gene (N) of 3 clone's IBV H120 vaccine strains
IBV nucleoprotein (N albumen) can combine with the IBV geneome RNA, and the enhanced virus genome is in intracellular stability, and the efficient of raising rescue virus.IBV H120 vaccine strain nucleoprotein gene (N) is cloned in pVAX1 (Invitrogen) carrier for expression of eukaryon; By the T7 rna polymerase promoter daughter nucleus heart sequence on the pVAX1 carrier; Produce the mRNA of N gene through in-vitro transcription, can be in transfectional cell transient expression nucleoprotein.
Whole genome sequence (GenBank accession number FJ807652) according to IBV H120 vaccine strain; Design the amplimer of this virus nucleoprotein gene (N); Primer sequence and the position (seeing table 3) in genome or eukaryon expression plasmid pVAX1 thereof: in the segmental upstream primer NS of N gene amplification, introduce Hind III restriction enzyme enzyme recognition site; Introduce XhoI restriction enzyme enzyme recognition site among the downstream primer NA, can cut rear clone to eukaryon expression plasmid pVAX1 at enzyme to guarantee N gene PCR amplified production; Primer NT7S and NT7A are used for obtaining the N gene from pVAXN recombinant plasmid PCR; Wherein the NT7S primer is positioned at the upper reaches of pVAX1 carrier T7 rna polymerase promoter daughter nucleus heart sequence; And the NT7A primer is positioned at the downstream of pVAX1 carrier MCS; And introduced 25 base T, to guarantee having T7 rna polymerase promoter daughter nucleus heart sequence at 5 ' end of N gene amplification product, 3 ' end has polyA tail structure.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Table 3 nucleoprotein gene (N) amplification primers
Annotate: underlined letter is the restriction enzyme enzyme recognition sequence; "
*" represent that primer sequence is arranged in the position of H120 genome; "
*" represent that primer sequence is arranged in the position of pVAX1 carrier.
With H120 strain virus cDNA is template, and primer NS in the employing table 3 and NA carry out the pcr amplification of nucleoprotein gene.High-fidelity enzyme PrimeSTAR HS DNAPolymerase (TaKaRa) is adopted in the PCR reaction, makes up the reaction system of 50 μ L according to product description.Reaction conditions is: 95 ℃, and 25s; 52 ℃, 25s; 72 ℃, 1min.The PCR product adopts Hind III and Xho I to carry out double digestion after 1% agarose gel electrophoresis detects.Enzyme is connected to equally and cuts in the pVAX1 carrier of processing through Hind III and Xho I enzyme after cutting the recovery of product glue, transforms the JM109 genetic engineering bacterium, cuts the evaluation positive colony through enzyme.The positive colony that obtains is served sea living worker's biotechnology Services Co., Ltd carry out sequencing, in full accord to confirm N gene cDNA fragment and the virus genome sequence inserted among the recombinant plasmid pVAXN.
Recombinant plasmid pVAXN with screening is a template, and primer NT7S and NT7A in the employing table 3 carry out pcr amplification.High-fidelity enzyme PrimeSTAR HS DNAPolymerase (TaKaRa) is adopted in the PCR reaction, makes up the reaction system of 50 μ L according to product description.Reaction conditions is: 95 ℃, and 25s; 52 ℃, 25s; 72 ℃, 1min.The PCR product is after 1% agarose gel electrophoresis detects; Reclaim the cDNA of N gene according to Agarose Gel DNA Fragment Recovery Kit (TaKaRa) working instructions glue; Its 5 ' end has T7 rna polymerase promoter daughter nucleus heart sequence, and 3 ' end has polyA tail structure.Measure its concentration, in-20 ℃ of preservations.
4 from H120 strain virus full-length gene group cDNA rescue virus
Get the nucleoprotein gene cDNA that the 1 H120 strain full-length gene group cDNA and 3 that makes up obtains; Adopt in vitro Transcription T7 in-vitro transcription test kit (TaKaRa); Carry out in-vitro transcription through the T7 promotor; The RNA cap structure analogue (NEB) that in the system of transcribing, adds simultaneously 2.5mM/ μ L makes and transcribes the RNA 5 ' end that obtains and have cap sequence.Make up the reaction system of 50 μ L according to product description, transcribe the DNase I that adds 2 μ L behind the 3h in 37 ℃ and handle 20min, the RNA of acquisition measures concentration through ultraviolet spectrophotometer.
Transfection previous day, the BHK-21 cell is passed on 6 orifice plates, in the high sugar of 10% DMEM (GIBCO) substratum, cultivate down for 37 ℃, when reaching 80%, cell density can carry out transfection when above.After the transcription product of above-mentioned H120-R full-length cDNA and nucleoprotein gene cDNA mixed according to 1: 5 concentration ratio, get 10 μ g blended RNA and join mixing in the 500 μ L RNase-free PBS solution; Get 30 μ L GenJet Plus (SignaGen) transfection reagents and be dissolved in mixing in the 500 μ L RNase-free PBS solution; GenJet Plus transfection reagent solution is joined in the above-mentioned RNA mixing solutions mixing immediately, incubated at room 15min～20min.During this period; With the BHK-21 cell in RNase-free PBS solution washing 6 orifice plates 3 times; Every then hole is got 200 μ L GenJet Plus/RNA mixtures and is joined on the BHK-21 cell of individual layer, remains to add 200 μ L RNase-free PBS solution in 1 hole as contrast.After placing 37 ℃ CO2 incubator to cultivate 5h, inhale and abandon transfection liquid, every hole adds 10% DMEM high glucose medium 2mL and continues to cultivate.After 48 hours, scrape the BHK-21 cell of getting on the culture plate, after nutrient solution and cell mixture freeze thawing 3 times, inoculate 10 age in days SPF chicken embryos.After placing 37 ℃ incubator to hatch 48h the inoculated into chick embryo, put 4 ℃ of refrigerator overnight, gather in the crops RT-PCR then and be accredited as the male chick embryo allantoic liquid, E1 is for kind of a poison in called after H120-R strain.H120-R (E1) is planted poison inoculate 10 age in days SPF chicken embryos once more, E2 is for kind of a poison in results chick embryo allantoic liquid called after H120-R strain.
H120-R (E2) is planted poison further adopts RT-PCR, digestion with restriction enzyme and sequencing to identify.Sequencing result (Fig. 5) shows that the base of the 738th of H120-R (E2) kind virus gene group is T, but not the C of its parent H120 strain conforms to the genetic marker result who sets; The Bsa I restriction enzyme enzyme recognition site at this place has been eliminated in sudden change, make the PCR product of H120-R (E2) after Bsa I digestion with restriction enzyme is handled, can not be cut off, and being cut off of parent H120 strain conforms to expected results.Sequencing result shows that the base of the 17243rd of H120-R (E2) kind virus gene group is T, but not the G of its parent H120 strain conforms to the genetic marker result who sets; A Bsa I restriction enzyme enzyme recognition site has been introduced in the sudden change at this place, handles with Bsa I digestion with restriction enzyme, and the PCR product of H120-R (E1) can be cut off, and the PCR product of parent H120 strain can not be cut off, and is consistent with expected results.Above result shows, through the reverse genetic manipulation technology, utilizes the H120 strain full-length gene group cDNA that makes up successfully to save and obtains to have infective progeny virus H120-R strain.
5 replace to the H120 strain full-length gene group cDNA rescue virus of EGFP gene from the 5a gene
The 5a gene of getting 2 structures replaces to the nucleoprotein gene cDNA that EGFP gene H120 strain full-length gene group cDNA and 3 obtains, and takes 4 identical methods to obtain the RNA product through in-vitro transcription, and mixes according to 1: 5 concentration ratio.Take 4 identical methods with RNA mixture transfection BHK-21 cell, after 48 hours, under the fluorescence inverted microscope,, can detect green fluorescence in the BHK-21 cell of part transfection through blue-light excited.
Scrape the BHK-21 cell of getting on 6 well culture plates, after nutrient solution and cell mixture freeze thawing 3 times, inoculate 10 age in days SPF chicken embryos.After placing 37 ℃ incubator to hatch 48h the inoculated into chick embryo, put 4 ℃ of refrigerator overnight, gather in the crops RT-PCR then and be accredited as the male chick embryo allantoic liquid, E1 is for kind of a poison in called after H120-5a/EGFP strain.H120-5a/EGFP (E1) is planted poison inoculate 10 age in days SPF chicken embryos once more, E2 is for kind of a poison in results chick embryo allantoic liquid called after H120-5a/EGFP strain.
H120-5a/EGFP (E2) is planted malicious evaluation, take H120-R in 4 (E2) to plant the identical method of poison, qualification result all conforms to expected results.Simultaneously, the H120-5a/EGFP recombinant virus can be in infected cells or chicken embryo expressing green fluorescent protein.Get chorioallantoic membrane that H120-5a/EGFP (E1) plants malicious inoculated into chick embryo, amnion etc. and organize compressing tablet, under the fluorescence inverted microscope after blue-light excited, all can detect a large amount of green fluorescence cells (Fig. 6).Above result shows, through the reverse genetic manipulation technology, utilizes the 5a gene that makes up to replace to EGFP gene H120 strain full-length gene group cDNA and successfully saves and obtain to have infective progeny virus H120-5a/EGFP strain.
6 detect the biological characteristics of rescue virus
Cause the test of short and smallization of chicken embryo: with the H120-R (E2) that obtains in 4 and 5 plant poison, H120-5a/EGFP (E2) plants malicious; After carrying out 1: 100 times of dilution with saline water; Be inoculated in respectively in 12 piece of 10 age in days SPF chick embryo allantoic cavity, 0.2mL/ piece, put 37 ℃ and take out 7 pieces of chicken embryos when hatching to 36h～48h; Place spend the night back results allantoic fluid, called after H120-R (E3) and H120-5a/EGFP (E3) respectively in 4 ℃ of refrigerators.Remain 5 pieces of chicken embryos and continue to hatch, observe the variation of chicken embryo to 144h.The result all can cause chicken embryo death after showing H120-R (E2) and H120-5a/EGFP (E2) infected chicken embryo, and characteristic change occurs: amnion thickens, and is close to idiosome; Chick embryo development is obstructed; Idiosome is curled than short and small with the normal chicken embryo of age in days, claims " dwarf embryo " (Fig. 7) again.It is identical with its parent's strain H120 to the pathogenic sex change of chicken embryo with H120-5a/EGFP to save viral H120-R.
The toxicity test of virus: get the kind poison of the strain H120-R (E2), H120-5a/EGFP (E2) and the parent's strain H120 that obtain in 4 and 5, do continuous 10 times with saline water respectively and be diluted to 10
-7Each dilution viral liquid is inoculated 5 pieces of 10 age in days SPF chicken embryos, 0.1mL/ embryo through allantoic cavity; Set up control group simultaneously, inoculate 5 pieces of 10 age in days SPF chicken embryos respectively with saline water.After the egg inoculation, put 37 ℃ of hatchings, abandon dead embryo in the 24h, every later at a distance from 12h observation 1 time, in time observe embryo development and pathology situation.Observed and recorded is pressed the EID that Reed～Muench Er Shi method is calculated virus to connecing poison back the 6th day
50The result shows the EID of strain H120, H120-R (E2), H120-5a/EGFP (E2)
50Be respectively 10
-6.84/ ML, 10
-6.83/ ML and 10
-6.38/ ML, the virulence of H120-R (E2) strain and the basically identical of parent's strain H120, and the virulence of H120-5a/EGFP (E2) strain is a little less than its parent's strain H120.
The hemagglutination activity of virus is measured: get H120-R (E2) the kind poison, the H120-5a/EGFP (E2) that obtain in 4 and 5 and plant poison, inoculate 10 age in days SPF chicken embryos respectively and carry out continuous passage, obtain its E3, E4, E5 and E6 for kind of a poison.Get above allantoic fluid of planting poison, be that the I type LecithinaseC (Sigma) of 1U/mL is handled 120min in 37 ℃ through final concentration, and after 4 ℃ of refrigerators placed for 2 weeks, carry out hemagglutination test by the micromethod operation.The result shows, each generation H120-R that handles without I type LecithinaseC and H120-5a/EGFP strain kind poison and parent's strain H120 thereof all do not have hemagglutination activity; After I type LecithinaseC is handled, E2～E6 of H120-R for the blood clotting price of kind of poison between 7log2～8log2, with the average blood clotting valency 7.6log2 basically identical of parent's strain H120; And E2～E6 of H120-5a/EGFP for the blood clotting price of kind of poison between 6log2～7log2.
The mensuration of viral growth curves: get the kind poison of the strain H120-R (E2), H120-5a/EGFP (E2) and the parent's strain H120 thereof that obtain in 4 and 5, with after the saline water dilution, inoculate each 25 pieces of 10 age in days SPF chicken embryos, 10 respectively through allantoic cavity
3EID
50/ embryo.Respectively at inoculation back 12h, 24h, 36h, 48h, 60h, 72h and 84h results allantoic fluid, adopt fluorescence quantitative RT-RCR to measure the virus titer of different time sections.The result shows that the virus titer of reverse genetic manipulation rescue strain H120-R and parent's strain H120 thereof reaches the climax behind inoculated into chick embryo 36h, descend then gradually; And the virus titer of reverse genetic manipulation rescue strain H120-5a/EGFP reaches climax (Fig. 8) behind inoculated into chick embryo 48h.
The present invention can carry out gene wherein on the basis of the above necessary processing, sudden change, gene insert disappearance, gene substitution etc., studying these phenotype, present situations that change organism has which kind of to influence, for the development of new vaccine provides solid foundation.
Claims (14)
1. IBV H120 vaccine strain reverse genetic operating system is characterized in that this system comprises:
1) the full-length cDNA molecule of structure RNA viruses, its 5 ' end of this full-length cDNA molecule has the core sequence of complete T7 rna polymerase promoter, and 3 ' end has polyA tail structure;
2) the encode cDNA sequence of nucleoprotein (N) of said IBV H120 vaccine strain of auxiliary cDNA, auxiliary cDNA, its 5 ' end has the core sequence of complete T7 rna polymerase promoter, and 3 ' end has polyA tail structure;
3) transfectional cell.
2. IBV H120 vaccine strain reverse genetic operating system according to claim 1 is characterized in that, the base of the 738th of genome sports T from C in the full-length cDNA molecule.
3. IBV H120 vaccine strain reverse genetic operating system according to claim 1 is characterized in that, the base of the 17243rd of genome sports T from G in the full-length cDNA molecule.
4. IBV H120 vaccine strain reverse genetic operating system according to claim 1; It is characterized in that; CDNA sequence two ends in a plurality of recombinant plasmids of structure full-length cDNA molecule have the restriction enzyme enzyme recognition site; To realize the intersegmental directed cloning of sheet, be used to make up the full-length cDNA molecule of said IBV H120 vaccine strain.
5. IBV H120 vaccine strain reverse genetic operating system according to claim 4 is characterized in that described restriction enzyme is Bsa I.
6. IBV H120 vaccine strain reverse genetic operating system according to claim 1 is characterized in that the 5a gene in the full-length cDNA molecule replaces to the green fluorescence gene.
7. according to any one reverse genetic operating system among the claim 1-6, it is characterized in that wherein said IBV is the H120 vaccine strain.
8. according to any one reverse genetic operating system among the claim 1-6, it is characterized in that wherein said transfectional cell is BHK-21.
9. a method of utilizing the reverse genetic operating system rescue IBV of above-mentioned each claim is characterized in that, carries out according to the following steps:
1) after being cut, said a plurality of recombinant plasmid enzymes connect into the full-length cDNA molecule;
2) with full-length cDNA and said auxiliary cDNA after in-vitro transcription becomes RNA, the host cell of cotransfection virus replication permission;
3) results host cell and supernatant, the inoculated into chick embryo allantoic cavity is rescued and is obtained virus.
10. according to the method for the reverse genetic operating system of claim 9 rescue IBV, it is characterized in that said 1) be that the template reverse transcription becomes cDNA with the H120 strain virus geneome RNA that extracts earlier in the step; Then said cDNA is carried out pcr amplification, the base mutation of 17243 of genomes in the amplification procedure; Be connected to the laggard row filter of pMD19-T carrier after the amplification, carry out the base mutation of the 738th of viral genome after the screening; Then inoculate the LB substratum and cultivate, reclaim the purpose fragment according to method then; Last each purpose fragment connects into the full-length cDNA molecule through restriction enzyme site.
11. save the method for IBV according to the reverse genetic operating system of claim 9; It is characterized in that; Said 2) the H120 strain virus geneome RNA with extraction is that the template reverse transcription becomes cDNA in the step; Carry out N protein gene pcr amplification then, then carry out Hind III and Xho I double digestion; The pVAX1 carrier carries out Hind III and Xho I double digestion equally simultaneously; Then both are connected and obtain recombinant plasmid pVAXN; PVAXN with screening is a template at last, reclaims the auxiliary cDNA that contains the N gene behind the pcr amplification.
12. the method according to the reverse genetic operating system of claim 10 is saved IBV is characterized in that, makes up in the full-length cDNA molecular process 5a gene is replaced to the green fluorescence gene.
13. the method for reverse genetic operating system rescue IBV according to claim 10; It is characterized in that; H120 pnca gene group is divided into 10 sections according to the distribution situation of IIs type restriction enzyme BsaI carries out pcr amplification; Then will according to the method in the claim 10 obtain purpose fragment TM1, TM2 ..., TM10, according to volumetric molar concentration 1: 1 TM2 is mixed with TM3, TM4 and TM5, TM6 and TM7, TM8 and TM9 at last and is connected, obtain intermediate product TM2-3, TM4-5, TM6-7, TM8-9; Then with TM1, TM2-3, TM4-5; TM6-7, TM8-9, TM10 connect according to 1: 1: 1 mixed of volumetric molar concentration respectively, obtain intermediate product TM1-5, TM6-10, TM1-5, TM6-10 are connected according to 1: 1 mixed of volumetric molar concentration to obtain the full-length cDNA molecule at last.
14. the method for reverse genetic operating system rescue IBV according to claim 13; It is characterized in that; Make up in the full-length cDNA molecular process 5a gene is replaced to the green fluorescence gene, all the other methods according to claim 14 obtain the full-length cDNA molecule.
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| CN107190022A (en) * | 2017-06-28 | 2017-09-22 | 浙江大学 | A kind of method of rapid build avian infectious bronchitis virus reverse genetic strain |
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