CN101220351A - Method for constructing PRRSV gene deletion vaccine toxin strain by using Nsp2 gene deletion and uses thereof - Google Patents

Abstract

The invention discloses a method for utilizing Nsp2 gene deletion for constructing a PRRSV gene deletion vaccine virus strain and the application thereof. The method for lowering the toxicity of the PRRSV BJ-4 strain of the invention is that a nucleotide segment with the length of 69nt in a Nsp2 coding region of the PRRSV BJ-4 strain genome is continuously deleted to obtain the virus strain with reduced toxicity; the nucleotide sequence of the 69nt nucleotide segment is shown as the 2795 to 2863 sites from the 5' tail end of GenBank Accession Number AF331831. The Nsp2 of the virus rV63 which is prepared by the method for lowering the toxicity of the PRRSV BJ-4 strain deletes 21 amino acids, which has the potential to develop a marker vaccine and can identify the diagnosis method of the deleted polypeptide. When in the development process of vaccine, the invention can utilizes a reverse genetic technical platform which is already established for carrying out the transformation of GP5 glycosylation sites, thus overcoming the suppression factors which affect the neutralizing epitope and further improving the level of the vaccine-induced neutralizing antibody.

Description

Utilize Nsp2 genetically deficient to make up the method and the application thereof of PRRSV gene-deleted vaccine strain

Technical field

The present invention relates to method and application thereof that a kind of Nsp2 of utilization genetically deficient makes up PRRSV gene-deleted vaccine strain.

Background technology

Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) be called as " pig is mysterious sick " at first, since phase late 1980s since the U.S. finds, be widely current in the country of respectively raising pigs, become global one of the most important diseases of industrial economy of raising pigs of harm.This sick principal character is kind of a pig breeding dysfunction, and the sucking piglets mortality ratio raises, the each age group pig particularly lactation and the child care stage piglet respiratory tract disease and the influenza-like symptom of growth and growing and fattening pigs.

Virulence factor as PRRS, porcine reproductive and respiratory syndrome virus (Porcine reproductive andrespiratory syndrome virus, PRRSV) at first be separated in the Holland and the U.S., and respectively as the prototype strain of Europe class (LV) and american type (VR-2332).The PRRSV genome is the sub-thread positive chain RNA, is about 15kb, and 5 ' end has cap sequence, and 3 ' end has poly (A) tail.Geneome RNA have 9 open reading frame (open-reading frames, ORFs).Two big ORF that are positioned at 5 ' end account for genomic 4/5ths, the replicative enzyme polyprotein of coding virus, and under the effect of shear protein enzyme, producing 13 kinds of Nonstructural Proteins, among all the other 7 ORF except ORF3 remains dispute, the viral structural protein of all encode.

PRRSV Nonstructural Protein 2 (Nsp2) has chymotrypsin-like (or microRNA virus 3C sample) L-Cysteine HCL Anhydrous structural domain, linear B cell epitope and potential t cell epitope.Infer that Nsp2 may be relevant with the assembling of the cell/tissue preferendum of virus and the shearing of species specificity, Nsp2/3 and Nsp4-8, two adipose membrane vesica (double lipid membranevesicles) and polyprotein virus replication combined enzyme agent (multiprotein viral replication complexes) etc.Yet except the B cell epitope, other function all is not proved.Still be amino acid levels analysis from Nucleotide no matter, Nsp2 is one of zone of PRRSV variability maximum.The variation of Nsp2 gene has point mutation, insertion and deletion mutantion, not only between special-shaped strain, occur, also be common in (Fang Y between the homotype strain, Kim D Y, Ropp S, et al.Heterogeneity in Nsp2 of European-like porcine reproductive andrespiratory syndrome viruses isolated in the United States.Virus Res, 2004,100 (2): 229 ~ 235).What relation is the chemical structure diversity of Nsp2 have it be unclear that with the biological function and the Clinical symptoms of virus.The reverse genetic technology provides a brand-new technology platform for molecular basis, protein function, virus and the host's of research the duplicating of RNA viruses, pathogenic, virulence difference interaction and development of new vaccine and virus vector.At present, 5 strain PRRSV strains having been arranged---the infections clone of LV, VR-2332, NVSL#97-7895, P129 and PL97-1 is succeedd.Yet follow-up research nearly all concentrates on the function of structural protein and non-coding region.Chen Xiaoyun etc. made up PRRSV BJ-4 strain full-length cDNA molecule (Chen Xiaoyun, Yang Hanchun, Guo Xin. the structure of porcine reproductive and respiratory syndrome virus BJ-4 strain full-length cDNA. Journal of Agricultural Biotechnology, 2005,13 (1): 61-65).

Summary of the invention

The method that the purpose of this invention is to provide a kind of PRRSV of reduction BJ-4 strain virulence.

The method of reduction provided by the present invention PRRSV BJ-4 strain virulence is that length is the nucleotide fragments of 69nt in the Nsp2 coding region of consecutive miss PRRSV BJ-4 pnca gene group, obtains the strain that virulence reduces; The nucleotide sequence of described 69nt nucleotide fragments as GenBank Accession Number AF331831 from 5 ' terminal the 2795th to 2863 shown in.

Insert foreign gene for the ease of reducing in the strain in resulting virulence, in the described method, length is on the basis of nucleotide fragments of 69nt in the Nsp2 coding region of described consecutive miss PRRSV BJ-4 strain, also can be again GenBankAccession Number AF331831 in 5 ' terminal the 2795th to 2863, insert restriction endonuclease sites, as the HpaI site.

The strain that above-mentioned any method obtains also belongs to protection scope of the present invention.

The strain that above-mentioned any method obtains can be used for preparation and prevents and/or treats the porcine reproductive and respiratory syndrome vaccine.

The present invention is by in-vitro transcription under the control of SP6 promotor and liposome-mediated direct transfection MARC-145 cell, not only obtained to stablize the wild-type that goes down to posterity and saved viral rV68, and made up successfully and obtained that length is the rescue virus rV63 of the nucleotide fragments of 69nt in the Nsp2 coding region of consecutive miss PRRSVBJ-4 pnca gene group.69nt disappearance not only can increase the ability that rV63 holds foreign gene, also makes to set up serology differential diagnosis method and become possibility; The unique site of HpaI of introducing also makes follow-up molecule manipulation convenient.So rV63 can become the ideal candidates object of PRRS marker vaccine and arteritis virus carrier.

The PRRSV of rescue clones viral rV63 and rV68 and has a good genetic stability external.PRRSVBJ-4 compares with parental virus, and wild-type is cloned viral rV68 duplicating and breed to some extent sluggishly on the MARC-145 cell, and the absence type of rescue is cloned viral rV63 and strengthened to some extent.Show that rV63 has stronger adaptation and multiplication capacity on the MARC-145 cell.

Zoogenetic infection test shows that rV68's is pathogenic lower than parental virus PRRSV BJ-4, and the virulence of rV63 has further and weakens.By these 21 amino acid of Nsp2 disappearance that reduce the viral rV63 of PRRSV BJ-4 strain virulence method preparation, have the potentiality that are developed to marker vaccine, and the polypeptide that can lack exploitation differential diagnosis method.In exploitation vaccine process, can utilize the reverse genetic technology platform of having set up that the glycosylation site of GP5 is transformed, overcome the damper that influences neutralizing epitope, to improve vaccine-induced neutralizing antibody level.

Description of drawings

Fig. 1 for primer P2327F and P2773R from the increase specific fragment of 486bp of plasmid pGEM-A

Fig. 2 is a pTE-nsp2d63 transformed bacteria bacterium colony PCR qualification result

Fig. 3 is EcoT22I and the HpaI single endonuclease digestion qualification result of pTE-nsp2d63

Fig. 4 is EcoT22I and the Nsb I single endonuclease digestion qualification result of pGEM-Am and pTE-nsp2d63m

Fig. 5 is that pGEM-Ad63 transformed bacteria bacterium colony PCR identifies

Fig. 6 is the excalation result of Nsp2 coding region in the pGEM-Ad63 plasmid

Fig. 7 is that pWSK-DCBAd63 transformed bacteria bacterium colony PCR identifies

Fig. 8 is connection site and a two side portions sequencing result thereof in the pWSK-DCBAd63 plasmid

Fig. 9 is that the 5th generation clone virus infects the back CPE that caused in 3 days (100 *) with parental virus

Figure 10 identifies for the indirect immunofluorescence of viral rV68 of clone and rV63

Figure 11 detects for the genetic marker of viral rV68 of clone and rV63

Figure 12 is the electron microscopic morphology of the negative staining clone virus particle of viral rV68 of clone and rV63

Figure 13 is that the partial sequence of rV63 (the 20th generation) and PRRSV BJ-4Nsp2 coding region compares

Figure 14 is clone's virus and the growth curve of parental virus on the MARC-145 cell

Figure 15 is the lungs cardinal principle pathology of rescue virus infection piglet

Figure 16 is the body temperature dynamic change of rescue virus infection piglet

Figure 17 is the ELISA antibody dynamic change of rescue virus infection piglet

Figure 18 is rescue virus infection porcine blood serum RT-PCR detected result

Figure 19 is that rV68 infected pigs isolated viral genetic marker detects

Figure 20 is that the isolated strain genetic marker order-checking of rV68 infected pigs is identified

Figure 21 is standard plasmid real-time fluorescence quantitative PCR result

Figure 22 detects for the antigenic immunohistochemical methods of rescue virus infection pig body inner virus

Embodiment

Embodiment 1, reduction PRRSV BJ-4 strain virulence obtain saving viral rV63

Used material among this embodiment:

A, cell and strain

MARC-145 cell (China Veterinary Drugs Supervisory Inst.) is used the DMEM culture medium culturing.PRRSV BJ-4 strain (China Agricultural University) in 1997 by Yang Hanchun (Yang Hanchun, Guan Shanhong, Yin Xiaomin, Deng. the separation of porcine reproductive and respiratory syndrome virus and preliminary evaluation. Chinese animal doctor's magazine, 1997,23 (10): 9-10) wait separation to preserve, its genome sequencing is finished (Yang Hanchun etc., 2001 (Yang Hanchun, Huang Fangfang, Guo Xin, etc. porcine reproductive and respiratory syndrome virus virus (PRRSV) BJ-4 strain whole genome sequence is measured and is analyzed. Journal of Agricultural Biotechnology, 2001,9 (3): 212-218) (the GenBank number of including is AF331831).

B, carrier, bacterial strain and PRRSV BJ-4cDNA recombinant plasmid

PGEM ^-T Easy Vector kit available from Promega company (Promega Corporation, WI, USA).

Recombinant plasmid pGEM-A contains the cDNA of PRRSV BJ-4 strain 1-4629nt.PWSK-DCBA contains PRRSV BJ-4 genome full-length cDNA, wherein introduces a unique Vsp I site as genetic marker.More than two recombinant plasmids make up (Chen Xiaoyun by the Ministry of Agriculture of China Agricultural University Preventive Veterinary Medicine emphasis open laboratory, Yang Hanchun, Guo Xin. the structure of porcine reproductive and respiratory syndrome virus BJ-4 strain full-length cDNA. Journal of Agricultural Biotechnology, 2005,13 (1): 61-65).These two recombinant plasmids can obtain from the Ministry of Agriculture of China Agricultural University Preventive Veterinary Medicine emphasis open laboratory.

PRRSV BJ-4 full length cDNA sequence is measured:

In order to prove conclusively the integrity and the reliability of the full gene of PRRSV BJ-4 among the pWSK-DCBA, carry out complete sequence determination with universal primer and PRRSVBJ-4 Auele Specific Primer.Sequencing result shows that the BJ-4 genome cDNA total length among the pWSK-DCBA is 15412bp (not containing poly (A)), there is a phage SP6RNA polymerase promoter core sequence in genome 5 ' upstream, many external source G that can promote in-vitro transcription efficient between first Nucleotide of promotor and viral genome.Genome 3 ' has 43 poly (A), before connecting than total length (pGEM-D) lack 40 (Chen Xiaoyun, Yang Hanchun, Guo Xin. the structure of porcine reproductive and respiratory syndrome virus BJ-4 strain full-length cDNA. Journal of Agricultural Biotechnology, 2005,13 (1): 61-65).In addition, compare with the sequence among the GenBank Accession Number AF331831, there are 3 Nucleotide that insert continuously its Nsp2 coding region.In addition, the point mutation of 23 stochastic distribution is arranged in full-length cDNA, wherein 11 are same sense mutation (contain and introduce Vsp I site mutation), and all the other 12 cause amino acid whose displacement, and they lay respectively at coding region and ORF4 and the ORF5 (table 1) of Nsp2, Nsp3, Nsp7 and Nsp9.

BJ-4 genome full-length cDNA among the table 1pWSK-DCBA and PRRSV BJ-4 genome sequence are relatively

The position a	Codeword triplet	Nucleotide changes	Mutation type	Amino acid change	Place albumen
						532	TCG	G→A	Replace	Reticent	Nsp1α
2413	AAT	T→G	Replace	N→K	Nsp2
						2823	CTG	T→C	Replace	L→P	Nsp2
3421	GCG	.→G	Insert	Insert	Nsp2
						3422	GCG	.→C	Insert	Insert	Nsp2
3423	GCG	.→G	Insert	Insert	Nsp2
						3482	TTG	T→A	Replace	L→F	Nsp2
3621	ACA	C→T	Replace	T→I	Nsp2
						3919	TCG	G→A	Replace	Reticent	Nsp2
4804	CCC	C→T	Replace	Reticent	Nsp3
						4838	CCC	C→T	Replace	P→S	Nsp3
4880	AGC	C→T	Replace	Reticent	Nsp3
						5011	TCC	C→T	Replace	Reticent	Nsp3
5288	CTG	C→T	Replace	Reticent	Nsp3
						7224	GCT	C→T	Replace	A→V	Nsp7
7277	GAG	G→A	Replace	E→K	Nsp7
						7479	AGG	G→A	Replace	R→K	Nsp7
8220 b	CCG	G→A	Replace	Reticent	Nsp9
						8372	CTT	T→C	Replace	L→P	Nsp9

8936	GGA	G→A	Replace	G→E	Nsp9
						9357	ATA	A→T	Replace	Reticent	Nsp9
11079	CCG	G→A	Replace	Reticent	Nsp11
						13472	TAT	T→C	Replace	Reticent	GP4
13623	ATC	A→G	Replace	I→V	GP4
						14055	ACG	G→A	Replace	Reticent	GP5
14110	TGT	T→A	Replace	C→S	GP5

^aNucleotide position refers to position (the GenBank number of including: AF331831) in PRRSV BJ-4 sequence.

^bVsp I is manually-injected genetic marker.

C, main molecules biological reagent

Easy A ^TMHigh-fidelity PCR cloning enzyme, QuikChange ^Multi site-directedmutagenesis kit available from Stratagene company (Stratagene, Aarhus, Denmark).EndoFree ^Plasmid maxikit available from QIAGEN company (Qiagen GmbH, Germany).MMessage mMachine ^HighYield Capped RNA Transcription kit and MEGA ^Clear kit available from Ambion company (Ambion Inc., Texas, USA).DMRIE-C (1,2-dimeyristyloxypropyl-3-dimethyl-hydroxy ethyl ammoniumbromide and cholesterol) and TRIzol ^LS available from Invitrogen company (Invitrogen lifetechnologies, CA, USA); GIBCO ^TMOpti-MEM ^I Reduced Serum Medium available from Invitrogen company (Invitrogene Corporation, NY, USA).Wizard ^Plus Midipreps DNA PurifationSystem, AMV ThermoScript II, Proteinase K, Vsp I are available from Promega company.Not I, Fse I, Sph I and T4DNA ligase enzyme available from NEB company (New England Biolabs (Beijing) Ltd, Beijing).EcoT22I, NsbI, HpaI, 15kb DNA marker are available from precious biotechnology (Dalian) company limited.RNase A available from Amresco company (AMRESCO Inc., Ohio, USA).DNA glass milk fast purifying reclaims test kit available from the vast Imtech in Beijing.DTT, RNsin, dNTPs, 100bp DNA Ladder and Taq archaeal dna polymerase are Time Inc. available from sky, Beijing.

D, other reagent

PRRSV N albumen McAb SDOW17 (Nelson E A, Christopher-Hennings J, Drew T, et al.Differentiation of U.S.and European isolates of porcine reproductive andrespiratory syndrome virus by monoclonal antibodies.J Clin Microbiol, 1993,31 (12): 3184-3189).

PRRSV BJ-4 strain GP5 albumen McAb F12F10 by the Ministry of Agriculture of China Agricultural University Preventive Veterinary Medicine emphasis open laboratory preparation (Yao Jiancong. porcine reproductive and respiratory syndrome virus BJ-4 strain GP5 Study of Monoclonal Antibodies: [master thesis]. Beijing: China Agricultural University, 2004).

Fluorescein (FITC)-conjugated AffiniPure rabbit anti-mouse IgG (H+L) is available from Jackson ImmunoResearch Laboratories company.

IPTG, X-gal, DEPC, agarose are available from Sigma company.Saturated Tris phenol is available from Beijing ancient cooking vessel state cell cultures solution

5 * DMEM or RPMI-1640 concentrated solution are with GIBCO ^TMDMEM or RPMI-1640 (USA) pulvis is pressed the shop instruction preparation for InvitrogenCorporation, NY, filtration sterilization, and 5 times of dilutions are carried out in 4 ℃ of preservations during use.

Foetal calf serum (FBS) Hyclone company (HyClone Laboratories Inc., UT, USA) product, 56 ℃ of water-bath deactivation 30min ,-20 ℃ of preservations are standby.

2 * 10 ⁴U/mL mycillin liquid (two anti-) penicillin, Streptomycin sulphate each 2 * 10 ⁶U is dissolved in the 100mL tri-distilled water, filtration sterilization, and after the packing ,-20 ℃ of preservations are standby.

7.5%NaHCO ₃7.5g NaHCO ₃Be dissolved in the 100mL tri-distilled water, through 121 ℃ of autoclaving 15min, normal temperature is preserved standby.

Growth medium (10%FBS) adds 50mL 5 * DMEM or RPMI-1640 solution in the 175mL tri-distilled water, 25mL FBS, and 2.5mL 2 * 10 ⁴U/mL mycillin liquid is used 7.5%NaHCO ₃Solution is regulated the pH value to the 7.2-7.4, and 4 ℃ of preservations are standby.

Keep substratum (2%FBS) and in the 195mL tri-distilled water, add 50mL 5 * DMEM or 5 * RPMI1640 solution, 5mL FBS, 2.5mL 2 * 10 ⁴U/mL mycillin liquid is used 7.5%NaHCO ₃Solution is regulated the pH value to the 7.2-7.4, and 4 ℃ of preservations are standby.

10 * Na ₂EDTA-pancreatin solution (2.5%) Na ₂EDTA 0.2g, NaCl 8.0g, KCl 0.2g, Na ₂HPO ₄12H ₂O2.89g, KH ₂PO ₄0.2g, be dissolved in the 90mL tri-distilled water, add 1: 250 pancreatin (Amresco Inc.) 2.5g again, after the dissolving, add tri-distilled water and complement to 100mL, NaHCO fully ₃Regulate pH to 7.2-7.4, filtration sterilization, packing ,-20 ℃ of preservations are standby, dilution in 1: 10 during use.

PBS(10mmol/L，pH7.4)

NaCl 8.00g

KH ₂PO ₄ 0.24g

Na ₂HPO ₄ 1.44g

KCl 0.20g

Add the 900mL tri-distilled water, heating is settled to 1000mL after dissolving fully, 121 ℃ of sterilization 15min.

The LB/Amp liquid culture is pressed final concentration 100 μ g/mL and is added penbritin based in the LB liquid nutrient medium.

LB/Amp solid medium agar powder 1.5g is dissolved among the 100mL liquid LB, and 115 ℃ of autoclaving 20min are cooled to 50 ℃, and adding penbritin to final concentration is 100 μ g/mL, and preparation is dull and stereotyped.

The LB/Amp/X-Gal/IPTG flat board adds 40 μ L X-Gal (20mg/mL) and 4ul IPTG (200mg/mL) on the LB/Amp flat board for preparing, evenly coat planar surface with sterilization L type glass rod, and 37 ℃ are incubated to absorption fully.

NYZ ⁺Liquid nutrient medium claims NA amine (casein hydrolysate) 10g, Yeast Extract 5g, and NaCl 5g with the distilled water dissolving, is settled to 1000mL, regulates pH to 7.5 with NaOH.Autoclaving, the 1mol/L MgCl of the cooling aseptic adding filtration sterilization in back ₂12.5mL, 1mol/L MgSO ₄12.5mL, 20% (20g/100mL) glucose 20mL, mixing gets final product.

Present embodiment is in the pWSK-DCBA that contains PRRSV BJ-4 genome full-length cDNA, and length is the nucleotide fragments of 69nt in the Nsp2 coding region of disappearance PRRSV BJ-4 pnca gene group, obtains recombinant vectors pWSK-DCBAd63; The nucleotide sequence of described 69nt nucleotide fragments as GenBank Accession Number AF331831 from 5 ' terminal the 2795th to 2863 shown in.The wild plasmid pWSK-DCBA and the pWSK-DCBAd63 of the non-disappearance of transfection evidence have infectivity, can save out virus.Rescue virus difference called after rV68 and rV63 from pWSK-DCBA and pWSK-DCBAd63 acquisition.Clone's virus rV68 of rescue is the same with parental virus BJ-4 with rV63, can cause the distinctive cytopathy of PRRSV (CPE) stable going down to posterity more than 20 generations on the MARC-145.The growth kinetics of deletion mutantion virus rV63 on the MARC-145 cell strengthens to some extent, and the incubation time that reaches high proliferation titre is cloned viral rV68 than parental virus PRRSV BJ-4 and wild-type respectively and shifted to an earlier date 12h and 24h.Concrete experimental technique and result are as follows:

One, the structure of recombinant vectors pWSK-DCBAd63

1, the structure that contains the recombinant vectors pTE-nsp2d63 of the 486bp nucleotide fragments Ad63 that is useful on disappearance Nsp2 coding region 69nt

According to PRRSV BJ-4 complete sequence (GenBank sequence number AF331831), with Oligo 6.0 software designs clone and detection primer (table 2-1), primer is synthetic by the living worker in Shanghai bio-engineering corporation.

PGEM-T Easy complete sequence (http://www.promega.com/vector/pgemtez.txt) and Stratagene company mutant primer design instruction according to Promega company, share rite-directed mutagenesis primer-design software (http://labtools.stratagene.com/QC) with the network that Stratagene company provides, design 35 ' phosphorylation primers (table 2), synthetic portion is synthetic by Shanghai Bo Ya Bioisystech Co., Ltd Beijing.

Table 2. is used for PRRSV BJ-4Nsp2 disappearance structure and detection and pGEM-T Easy carrier rite-directed mutagenesis primer sequence and position thereof

Primer title Primer	Sequence (5 ' → 3 ') a	The position b	Purpose
				P2327F	ACC GCC TTT TCA CTG GCT AAC TAC	2327-2350	Clone, detection
P2773R	GAA GAT GCA TTG GTT AAC GCT ACT AAC AGC CAA ATC TTC C(Hpa I)	2773-2812	The clone
				P2997R	CCG CCG AAC TCA ATC TTT TCA CCT	2997-3020	Detect

DetcM1	GAA CAA GTT TAA GGA GCT ACA GAC	8817-8841	Detect
				DetcM2	ATT GGA CCT GAG TTT TTC CCA CAT	9586-9610	Detect
TE127m	TCC CAA CGC GTT GGA TAC ATA GCT TGA GTA TTC TAA	109-143	Rite-directed mutagenesis
				TE1632m	TTC GCC AGT TAA TAG TTT CCG CAA CGT TGT TGC CA	1613-1647	Rite-directed mutagenesis
TE2855m	CGC CAT TCA GGC TGG GCA ACT GTT GGG AA	2841-2869	Rite-directed mutagenesis
				F1	TCG GAC GGT AAA AAG AAA AG	3829-3848	Detect
G2	GCC AAA GAG AAA CCA ACA AC	5404-5423	Detect

^aThe restriction enzyme site (be marked in bracket in) of horizontal line place for introducing, thickened portion is the base of sudden change, mutant primer 5 ' phosphorylation.

^bClone and detection primer location are meant position (the GenBank sequence number: AF331831) in the BJ-4 genome; The position of rite-directed mutagenesis primer is meant position (the Promega company: http://www.promega.com/vector/pgemtez.txt) in pGEM-T Easy carrier.

With the pGEM-A plasmid DNA is template, uses Easy A ^TMHigh-fidelity PCR cloning enzyme high-fidelity DNA polymerase and primer P2327F and P2773R (table 1) carry out.Add following reagent, set up 50 μ L reaction systems.

10×PCR buffer 5μL

dNTPs(10mmol/L，2.5mmol/L each) 2μL

P2327F(25μmol/L) 1μL

P2773R(25μmol/L) 1μL

PGEM-A (about 0.2g/L) 0.5 μ L

ddH ₂O 40μL

Easy A ^TM enzyme(5U/μL) 0.5μL

The PCR loop parameter is: 94 ℃ of 3min, and 94 ℃ of 30s, 58 ℃ of 40s, 72 ℃ of 45s, after 30 circulations, 72 ℃ of 10min.Get 5 μ L PCR products, check the pcr amplification result with 1.2% agarose gel electrophoresis.The result shows that with primer P2327F and P2773R from the increase specific fragment of 486bp of plasmid pGEM-A, name is called Ad63 (Fig. 1), and size conforms to design.Among Fig. 1, M:DNA molecular weight standard (100bp DNA Ladder); 1:P2327F and P2773R amplification.

Separate the PCR product with 1% agarose gel electrophoresis, downcut the purpose band, reclaim test kit with glass milk and reclaim.The PCR product is connected with pGEM-T Easy, obtains recombinant vectors pTE-nsp2d63.With pTE-nsp2d63 transformed into escherichia coli DH5 α competent cell, on the LB/Amp/X-gal/IPTG flat board according to blue hickie primary dcreening operation recombinant conversion bacterium.Select white colony, adopt previous reaction system (polysaccharase is changed to general T aq archaeal dna polymerase) and reaction conditions to carry out pcr amplification, get 5 μ L PCR product electrophoretic examinationss.Bacterium colony PCR result obtains the band of 486bp as shown in Figure 2, the positive reorganization of selected hickie bacterium.Among Fig. 2,1-6:pTE-nsp2d63 transforms bacterium colony; 7: negative control; 8:DNA molecular weight standard (100bp DNA Ladder).

This PCR positive bacteria is carried out single endonuclease digestion with EcoT22 I and Hpa I respectively to be identified.Cut the result with 1.2% agarose gel electrophoresis inspection enzyme.The enzyme of pTE-nsp2d63 is cut qualification result as shown in Figure 3, shows that EcoT22 I enzyme cuts pTE-nsp2d63 and obtain a linearizing band, and Hpa I enzyme is cut pTE-nsp2d63 and obtained a linearizing band.Among Fig. 3,1:pTE-nsp2d63 (EcoT22 I enzyme is cut); 2:pTE-nsp2d63 (Hpa I enzyme is cut); 3:pTE-nsp2d63 (not carrying out enzyme cuts); M1:15kb dna molecular amount standard; M2:100bp gradient dna molecular amount standard.

PTE-nsp2d63 is served the sea give birth to worker bio-engineering corporation, adopt the carrier universal primer to carry out complete sequence determination inserting fragment.The sequencing result of recombinant plasmid pTE-nsp2d63 shows that insertion sequence is the part of BJ-4 genome sequence, but at 2413 corresponding to BJ-4, a point mutation (T → G) is arranged, through finding relatively that with the pGEM-A sequence this point sudden change exists in template plasmid, be not to come from the PCR process.

2, EcoT22 I in the carrier sequence of pGEM-A and pTE-nsp2d63 plasmid and Nsb I are destroyed structure pGEM-Am and pTE-nsp2d63m

Find that through the DNAMAN software analysis pGEM-T Easy multiple clone site both sides exist EcoT22 I and Nsb I site, hinder next step subclone.Therefore, use QuikChange earlier ^Multi site-directed mutagenesis kit and mutant primer (table 1) destroy EcoT22 I in the carrier sequence of pGEM-A and pTE-nsp2d63 plasmid and Nsb I.

Add various compositions in the following order successively, set up 25 μ L reaction systems.Mixing, instantaneous centrifugal laggard performing PCR amplification.The PCR loop parameter of rite-directed mutagenesis is: 95 ℃ of 1min, 95 ℃ of 1min, 55 ℃ of 1min, 65 ℃ of 15min, totally 30 circulations.

10×QuikChange ^Multi reaction buffer 2.5μL

ddH ₂O 1μL

QuikSolution 0.75μL

ds-DNA template(100ng/μL) 1μL

TE127m(100ng/μL) 1μL

TE1632m(100ng/μL) 1μL

TE2855m(100ng/μL) 1μL

dNTPs 1μL

QuikChange ^Multi enzyme blend 1μL

The elimination of double-stranded template DNA.PCR reaction directly adds 1 μ L Dpn I (10U/ μ L) in the amplified reaction pipe after finishing, and with micro sample adding appliance pressure-vaccum gently, fully mixing is instantaneous centrifugal, 37 ℃ of water-bath 1h, digestion double-stranded template DNA.

From-80 ℃ of super competent cells of taking-up XL 10-Gold, place on ice and melt, divide the aseptic Eppendolf centrifuge tube of 1.5mL that installs to precooling, every 45 μ L behind the mixing.Add 2 μ L β-ME, behind the mixing, mixing is once gently for ice bath 10min, every 2min.

In above-mentioned competent cell, add the single stranded DNA that 1.5 μ L handle through Dpn I respectively, gently ice bath 30min behind the mixing.

42 ℃ of accurate heat shock 30s, ice bath 2min immediately.

Add and be preheating to 42 ℃ NYZ ⁺ Liquid nutrient medium 500 μ L, 37 ℃, 225r/min concussion cultivation 1h.

Get an amount of bacterium liquid coating LB/Amp flat board, 37 ℃ of overnight incubation (＞16h).Get the mutant plasmid DNA of pGEM-A and pTE-nsp2d63, carry out single endonuclease digestion with EcoT22I, Nsb I respectively and identify.Cut the result with 0.7% agarose gel electrophoresis inspection enzyme.Distinguish called after: pGEM-Am and pTE-nsp2d63m with identifying two kinds of plasmids that suddenly change successfully.The enzyme of pGEM-Am and pTE-nsp2d63m is cut qualification result as shown in Figure 4, pGEM-Am obtains a single linear dna molecular through EcoT22 I and Nsb I single endonuclease digestion respectively, pTE-nsp2d63m obtains a single linear dna molecular through EcoT22 I and Nsb I single endonuclease digestion respectively, show that EcoT22 I and Nsb I site in the carrier are destroyed, each keeps the unique site in the BJ-4cDNA fragment.Among Fig. 4,1 is the EcoT22 I single endonuclease digestion of pGEM-Am, and 2 is the EcoT22I single endonuclease digestion of pTE-nsp2d63m, and 3 is the Nsb I single endonuclease digestion of pGEM-Am, and 4 is the Nsb I single endonuclease digestion of pTE-nsp2d63m, and M1 is the 15kbDNA molecular weight standard; M2:100bp gradient dna molecular amount standard.

3, the structure that contains the recombinant vectors pGEM-Ad63 of the 486bp nucleotide fragments Ad63 that is useful on disappearance Nsp2 coding region 69nt

PGEM-Am and pTE-nsp2d63m are carried out EcoT22 I enzyme respectively to be cut.After enzyme cut mixture and show that with 1% agarose gel electrophoresis enzyme is cut thoroughly, add the NaAc (pH 5.2) of 5 μ L 3moL/L and the cold ethanol of 2 times of volumes ,-20 ℃ precipitate 1h; 4 ℃, the centrifugal 10min of 12000r/min abandon supernatant, and precipitation is washed once with 75% ethanol; Air-dry back adds 39 μ L sterilization ddH ₂The O dissolving.Whole EcoT22 I enzymes are cut product to carry out Nsb I enzyme and cuts.Enzyme is cut product with 1% agarose gel electrophoresis, downcut the purpose band, carry out purifying, recovery with glass milk by preceding method.The recovery product (486bp) of pGEM-Am and the recovery product of pTE-nsp2d63m (the big fragment of carrier) are connected.To connect product and transform the TOP10 competent cell respectively, coating LB/Amp+ flat board, 37 ℃ of incubator overnight incubation.

According to preceding method, respectively select some bacterium colonies, carry out bacterium colony PCR screening with primer P2327F, P2997R (table 1) and general T aq archaeal dna polymerase.The PCR loop parameter is: 94 ℃ of 3min, and 94 ℃ of 30s, 58 ℃ of 40s, 72 ℃ of 45s, after 35 circulations, 72 ℃ of 10min.Get 5 μ L PCR products, check the pcr amplification result with 1.5% agarose gel electrophoresis.The result connects the specific fragment that product amplifies about 480bp, and pGEM-Am amplifies the fragment (Fig. 5) of about 550bp size.Show that the Segment A d63 that contains Nsp2 coding region excalation successfully replaces the corresponding sequence among the pGEM-A.Pcr amplification is gone out the recombinant plasmid called after pGEM-Ad63 of the specific fragment of about 480bp.Among Fig. 5,1:100bp gradient dna molecular amount standard; 2:pGEM-A; 3, negative control; 4-7:pGEM-Ad63.

For the further clear and definite disappearance accurately that whether produces, the positive recombinant plasmid pGEM-Ad63 that PCR is identified carries out part order-checking evaluation.The result proof Nsp2 coding region in pGEM-Ad63 successfully lacks 69nt (GenBank AccessionNumber AF331831 from 5 ' terminal the 2795th to 2863), introduce a HpaI site (GTTAAC) simultaneously, the few 63nt (Fig. 6) of actual specific BJ-4.Among Fig. 6, A:pGEM-A; B:pGEM-Ad63; 7; Numeral: the position of indication Nucleotide in the BJ-4 genome.

4, in the pWSK-DCBA that contains PRRSV BJ-4 genome full-length cDNA, length is the nucleotide fragments of 69nt in the Nsp2 coding region of consecutive miss PRRSV BJ-4 pnca gene group, obtains recombinant vectors pWSK-DCBAd63

Plasmid pWSK-DCBA and pGEM-Ad63 carry out single endonuclease digestion respectively with Fse I earlier.After enzyme is cut mixture and confirmed complete linearizing with 0.7% agarose gel electrophoresis, carry out Not I enzyme more respectively and cut.Enzyme is cut mixture with 0.7% agarose gel electrophoresis, downcut long segment band and the pGEM-Ad63 short-movie section band that through double digestion after obtain of pWSK-DCBA respectively, connect through obtaining behind the double digestion.To connect product and transform DH5 α competent cell.Choosing colony carries out bacterium colony PCR screening with primers F 1 (being positioned at the A fragment), G2 (being positioned at the B fragment) (table 1) and general T aq archaeal dna polymerase.The PCR loop parameter is: 95 ℃ of 3min, and 94 ℃ of 30s, 56 ℃ of 40s, 72 ℃ of 100s, after 35 circulations, 72 ℃ of 10min.Get 5 μ L PCR products, check the pcr amplification result with 1% agarose gel electrophoresis.The result amplifies the specific fragment (Fig. 7) of about 1.6kb, shows that Ad63 replaces the A fragment among the pWSK-DCBA, obtains recombinant plasmid pWSK-DCBAd63.Among Fig. 7,1-2:pWSK-DCBAd63; 3:DNA molecular weight standard (100bp DNA Ladder); 4: negative control.

PCR is screened the male bacterium colony, inoculation 3mL LB/Amp liquid nutrient medium, 150r/min concussion overnight incubation under 28 ℃ of conditions.Afterwards, the ratio in 1/500 is seeded to 100mL LB/Amp liquid nutrient medium, 150r/min enlarged culturing 12h under 28 ℃ of conditions.With Wizard ^Plus Midipreps DNA Purifation System extracts and plasmid DNA purification.The by specification method is carried out.The plasmid of purifying is served the sea give birth to worker bio-engineering corporation, two connection site and both sides sequence thereof and Nsp2 coding region are carried out the part order-checking.Cover two connection site and two side portions sequence sequencing result (Fig. 8) thereof among the pWSK-DCBAd63, confirm with Nsp2 part sequencing result, pWSK-DCBAd63 is on the basis of pWSK-DCBA, length is the nucleotide fragments of 69nt in the Nsp2 coding region of consecutive miss PRRSV BJ-4 pnca gene group, and does not all introduce new sudden change near connection site and the deletion sequence.The nucleotide sequence of described 69nt nucleotide fragments as GenBankAccession Number AF331831 from 5 ' terminal the 2795th to 2863 shown in.

Two, obtain to save viral rV68 and rV63 respectively from pWSK-DCBA and pWSK-DCBAd63

Will be from linearization plasmid pWSK-DCBA, pWSK-DCBAd63, pWSK-DCBAd117 in-vitro transcription and the RNA that comes with liposome direct transfection MARC-145 cell.Because CPE does not all appear in the first-generation, cultivates after 5 days, get the continuous passage of transfection supernatant inoculation MARC-145 cell monolayer, per generation 5-7 days.Up to the 4th generation, still do not observe CPE.Transcribe and the RNA cells transfected supernatant that comes when reaching for the 5th generation by pWSK-DCBA and pWSK-DCBAd63, tangible CPE appears in the MARC-145 cell, present focal cell aggregation at first, become circle, shrinkage, break away from cell monolayer gradually, last disintegration, (Fig. 9) comes off.After infection 5-6 days, whole cell monolayer completely destroy, most of cell detachment.For further confirming to have virus-free low-level duplicating, its culture supernatant is carried out RT-PCR detect.The result only detects viral nucleic acid from the 1st generation, and during with the DetcM1 reverse transcription PCR, does not but detect viral strand RNA, and prompting is virus replication not.Show that the pWSK-DCBAd63 of structure has infectivity, can save out virus.Rescue virus difference called after rV68 and rV63 from pWSK-DCBA, pWSK-DCBAd63 acquisition.

Concrete experimental technique is as follows:

1, RNA in-vitro transcription and purifying

PWSK-DCBA and pWSK-DCBAd63 are digested with Sph I restriction endonuclease respectively, make the complete linearizing of closed loop plasmid DNA.Enzyme is cut product carry out 0.7% agarose gel electrophoresis detection, detected result shows pWSK-DCBA and pWSK-DCBAd63 complete linearizing respectively.MMessage mMachine according to Ambion company ^High YieldCapped RNA Transcription kit specification sheets carries out in-vitro transcription.MEGA according to Ambion company ^Clear kit specification sheets carries out the RNA purifying.

2, transfection

Select normal, the eugonic MARC-145 cell of form, after the trysinization, with about 10 ⁶The cell density of individual/mL changes six porocyte culture plates over to, every hole 2mL.37 ℃, 5%CO ₂, overnight incubation under the saturated humidity, until the cell monolayer that forms about 80%.Careful sucking-off cells and supernatant, every hole adds the Opti-MEM of 2mL balance to room temperature ^I ReducedSerum Medium washs 1-2.In the 1.5mL of no RNase eppendorf pipe, add the Opti-MEM of 1mL balance to room temperature ^I Reduced Serum Medium.With DMRIE-C liposome vortex mixing, get 10 μ L and add in each pipe, and mixing.In each pipe, add 5 μ g RNA respectively, slightly vortex mixing momently.Change liposome-RNA mixture over to cell monolayer immediately, establish Opti-MEM simultaneously ^I Reduced Serum Medium and the contrast of DMRIE-C liposome.CO ₂Behind 37 ℃ of cultivations of incubator 4h, careful sucking-off transfection liquid adds the 2mL growth medium.Cultivate after 5 days, CPE all do not occur, get the MARC-145 cell that culture supernatant inoculation forms 80% individual layer, continuous passage, per generation 5-7 days, and observation of cell pathology effect (CPE) day by day.Up to the 4th generation, still do not observe CPE.Transcribe and the RNA cells transfected supernatant that comes when reaching for the 5th generation by pWSK-DCBA and pWSK-DCBAd63, tangible CPE appears in the MARC-145 cell, present focal cell aggregation at first, become circle, shrinkage, break away from cell monolayer gradually, last disintegration, (Fig. 9) comes off.After infection 5-6 days, whole cell monolayer completely destroy, most of cell detachment.

3, the indirect immunofluorescence of clone's virus is identified

Got for the 5th generation (the 1st generation that CPE occurs) cells and supernatant, inoculation MARC-145 cell, fixing after 3 days, use at PRRSV N albumen and the proteic monoclonal antibody specific of GP5 according to reference literature (Yang Hanchun, Huang Fangfang, Zhang Xu, etc. the porcine reproductive and respiratory syndrome virus strain isolated identified with monoclonal antibody.China animal doctor magazine, 1998,23 (4): 3-5; Yao Jiancong. porcine reproductive and respiratory syndrome virus BJ-4 strain GP5 Study of Monoclonal Antibodies: [master thesis].Beijing: China Agricultural University, 2004) method is carried out indirect immunofluorescence and is detected, and compares with parental virus simultaneously.The result shows, in the endochylema of two viral rV63 of clone and rV68 and parental virus PRRSV BJ-4 cells infected, all present specificity fluorescent, and control cells (the MARC-145 cell of untransfected) no specificity fluorescent (Figure 10), show thus PRRSV specificity N is arranged in pWSK-DCBA and the pWSK-DCBAd63 transfectional cell, GP5 albumen is synthetic, determine to have obtained the infectious virus particle from the full-length cDNA that makes up.Among Figure 10, A:N albumen McAb SDOW17 dyeing, B:GP5McAbF12F10 dyeing; A:rV63; B:rV68; C:BJ-4; D: normal cell contrast (200 *).

4, the genetic marker of clone's virus detects

Owing to when making up infectious CDNA, in particular sequence, introduced specific genetic marker-VspI and HpaI restriction enzyme site, and in the corresponding sequence of parent's strain PRRSV BJ-4, do not had this mark.Extract viral RNA from the 5th generation cells and supernatant, the enzyme that carries out RT-PCR and specificity genetic marker with primer DetcM1 and DetcM2 (covering the VspI site in clone's virus) and P2327F and P2997R (covering the Hpa I site that lacks in the virus) (table 2) is cut evaluation respectively.The result is with all can increase from save virus (rV63 and rV68) and the parental virus fragment of 793bp size of primer DetcM1 and DetcM2, and can be cut into 540bp and 253bp two bands by Vsp I from saving the product that virus (rV63 and rV68) obtains, but the PCR product of parental virus can not be by Vsp I digestion (A among Figure 11).With all can increase from wild-type is cloned the culture supernatant of viral rV68 and the parental virus BJ-4 fragment of 695bp size of primer P2327F and P2997R, its product can not be cut by Hpa I; And clone can the increase fragment of 632bp size of viral rV63 from absence type, can be cut into 471bp, 161bp two bands (B among Figure 11) by Hpa I.A is that the Vsp I enzyme of primer DetcM2 and DetcM1 RT-PCR product is cut evaluation among Figure 11; B is that the Hpa I enzyme of primer P2997R and P2327F RT-PCR product is cut evaluation among Figure 11.

Show directly checking order behind the PCR product purification, the genetic marker of clone virus rV63, the rV68 of rescue and the disappearance of rV63 Nsp2 coding region (length is the nucleotide fragments of 69nt in the Nsp2 coding region of consecutive miss PRRSV BJ-4 pnca gene group, the nucleotide sequence of this 69nt nucleotide fragments as GenBank Accession Number AF331831 from shown in 5 ' terminal the 2795th to 2863) exist really.The above results has proved that further the 2 strain clone viruses (rV63 and rV68) of rescue are derived from infectious CDNA, have got rid of parental virus PRRSV BJ-4 contamination of heavy.

5, the morphological observation of clone's virus

The external continous-stable of clone virus that CPE occurs is passed 5 generations (i.e. the 10th generation), behind the ultracentrifugation, usefulness PRRSV antiserum(antisera) (with the PRRSV cell toxicant through 0.01% formaldehyde, 37 ℃ of deactivations of spending the night.Select 1 of SPF pig, two all immunity once are total to immunity 4 times at interval.Initial immunity adds Freund's complete adjuvant, and dosage is 1mg.Increase progressively 0.5mg later at every turn.Immunity for the second time adds Freund's incomplete adjuvant.Subcutaneous and the muscle branch injection of neck.The 4th immunity blood sampling in back 10 days, separation of serum, obtain the PRRSV antiserum(antisera)), according to document (Petit M A, Lievre M, Peyrol S, et al.Enveloped particles in the serum of chronic hepatitis C patients.Virology, 2005,336:144-153) method of Miao Shuing is carried out the immuno-electron microscope detection.Show that clone's virus particle of rescue is the representative configuration feature of PRRSV, and the spherical virus particle of cyst membrane is promptly arranged, size is 60-80nm (Figure 12).Among Figure 12, A:rV68; B:rV63; Scale: 50nm (600,000 times).

6, the clone of rescue virus cultural characters analysis

(1) TCID ₅₀Measure

According to document (Yin Zhen, Liu Jinghua. animal virology (second edition).Beijing: Science Press, 1997,329-331) method is got clone's virus of different generations, measures the malicious valency of virus on the MARC-145 cell.Concrete grammar is as follows:

Behind the MARC-145 cell dissociation, with 10 ⁶The density of individual/mL changes 96 porocyte culture plates over to, every hole 100 μ L.37 ℃, 5%CO ₂, overnight incubation under the saturated humidity, form about 80% cell monolayer, the careful suction abandoned supernatant.Add the careful washing of PBS once.Virus is carried out 10 times and increased progressively dilution continuously to keep substratum, get 10 ^-4-10 ^-8Dilution virus inoculation MARC-145 cell monolayer, each gradient 4 hole, every hole 100 μ L, CO ₂37 ℃ of absorption of incubator 1h.The careful suction abandoned supernatant, after the PBS washing once, adds 100 μ L and keeps substratum continuation cultivation.After 2 days, observe CPE day by day,, write down the hole count of each gradient CPE until infecting back 5 days.Calculate TCID according to Reed-M ü ench method ₅₀

(2) stability analysis of genetic marker

To on the MARC-145 cell, cultivate the 20th generation virus, 4 method is carried out RT-PCR, enzyme and is cut and identify and order-checking set by step, detects whether stable existence of genetic marker.

(3) growth curve is measured

Reference literature (Yuan S, Mickelson D, Murtaugh M P, et al.Erratum to " Complete genomecomparison of porcine reproductive and respiratory syndrome virus parental andattenuated strains " .Virus Res, 2001,79 (1-2): 189-200) method, get the 13rd generation parental virus and clone virus carry out.Concrete grammar is as follows: will plant malicious multigelation 3 times, 4 ℃, the centrifugal 30min of 3000r/min abandon cell debris, and it is standby to collect supernatant.Measure the malicious valency of supernatant on the MARC-145 cell, (0.01mol/L, pH7.2) dilution is 10 with PBS ⁵TCID ₅₀/ mL.Bottle (25cm with overnight incubation (about 80% individual layer) ²) the MARC-145 cell is with after the PBS washing, adds the virus of 1mL dilution respectively, 37 ℃ of absorption 1h.Discard virus inoculation liquid, after the PBS washing, respectively add 5mL and keep substratum continuation cultivation.Take out 200 μ L culture supernatant in different time, and in the cell bottle, add the substratum of keeping of same amount.Measure the TCID of each time point culture supernatant ₅₀Value is drawn growth curve.

The result that goes down to posterity on the MARC-145 cell shows that 2 strain clones virus rV68, rV63 passed for 20 generations continuously on the MARC-145 cell, and cytopathy is stable.After the 6th generation, the time that rV68 produces typical CPE is always after the infection about 84h, than the late 24h of parental virus PRRSV BJ-4; And rV63 causes the time of typical CPE about infecting back 60h, and is similar to parental virus.Examine under a microscope, parental virus PRRSV BJ-4 and the rV63 pathology due to MARC-145 is slightly more serious than rV68, and the time of cell detachment, early the area that comes off was big.

Be to understand the variation of crohn poison malicious valency after continuous passage on the MARC-145 cell, to the 5th, 9,13,19 generation culture carry out TCID ₅₀Measure, repeat 3 times, average malicious valency is as shown in table 3.Except that the 5th generation, each malicious valency of cloning between the viral different generation does not have significant difference (P＞0.05).Though the corresponding rV68 of each malicious price ratio of generation of rV63 poison valency is slightly high, find except the 5th generation difference not significantly (P＞0.05) between other each generation through t check carrying out statistical analysis.Show that clone's virus continuous passage poison valency on the MARC-145 cell is stable, the propagation titre does not have considerable change.

Malicious valency (the Log10TCID of the different generation clone of table 3 virus ₅₀/ mL)

Virus	The 5th generation	The 9th generation	The 13rd generation	The 19th generation
					rV68	4.67±0.19	7.50±0.20	7.00±0.23	7.23±0.4
rV63	6.00±0.21	8.23±0.23	7.83±0.17	8.15±0.17

Clone's virus the 20th generation poison of saving being carried out the enzyme of RT-PCR and product thereof cuts and sequential analysis (Figure 13), the result shows, in 2 clone's viruses among corresponding genetic marker and the rV63 disappearance still exist, show the sudden change of artificial introducing can be with viral subculture in vitro separately genetic stability.Among Figure 13, thickened portion is Hpa I site; Hyphen is represented identical; Round dot is represented disappearance.

The 13rd generation culture the growing on the MARC-145 cell of cloning virus increased dynamically and measure, and compare with parental virus, result (Figure 14) shows, after viral rV63 infection MARC-145 cell is saved in the Nsp2 deletion mutantion, 48h can reach high proliferation titre, and the virus titer in the culture supernatant of 36h, 48h, 60h is higher than parental virus PRRSV BJ-4, has significant difference (P＜0.05), the growth curve of the two is quite similar, is 60h but parental virus reaches the time of high proliferation titre.And rV68 is before 72h after the infection, the culture supernatant of collecting, its virus titer all significantly is lower than rV63 and parental virus PRRSV BJ-4 (P＜0.05), the time that reaches the titre peak value also postpones about 12h, 24h respectively than parental virus PRRSV BJ-4 and rV63, illustrates that the rate of propagation of clone virus rV68 on the MARC-145 cell of rescue is sluggish to some extent.The on average the highest titre of rV63, PRRSV BJ-4 and rV68 is respectively 10 ^9.47TCID ₅₀/ mL, 10 ^8.92TCID ₅₀/ mL, 10 ^8.52TCID ₅₀/ mL does not have significant difference (P＞0.05).

Though disappearance 21 amino acid in the variable region of rV63 Nsp2 are compared with parental virus with rV68, its growth on the MARC-145 cell strengthens to some extent.From growth curve as can be known, though the highest malicious valency of rV63 and all the other two strain virus does not have significant difference (P＞0.05), the time shifts to an earlier date more than the 12h.These results show that rV63 adapts to the MARC-145 cell more than BJ-4 and rV68.

Three, Zheng Jiu PRRSV clones viral rV68 and the rV63 pathogenic analysis to piglet

(Porcine reproductive and respiratory syndrome virus, variation phenomenon PRRSV) is commonplace for porcine reproductive and respiratory syndrome virus.Nsp2 is one of albumen of tool variability, and amino-acid substitution, disappearance and insertion all occur in the natural separation strain, and the virulence between the different isolated strain there are differences.For the deletion mutantion virus of analyzing rescue and parent clone virus pathogenic to piglet, to clone viral rV68 and rV63 inoculation piglet, record and observation clinical manifestation, cardinal principle pathology, day weight gain, viremia, serum antibody dynamic change, nasal cavity toxin expelling and virus antigen distribution situation.Two strain virus all only cause slight fervescence, upper respiratory tract infection and interstitial pneumonia; Do not detect the nasal cavity toxin expelling; The average daily gain of three treated animals does not have significant difference (P＞0.05).Infected group has 50% animal ELISA antibody commentaries on classics sun at 14DPI, all changes sun during 21DPI, and the antibody horizontal of the two does not have significant difference (P＞0.05); Duration of test does not detect neutralizing antibody.From rV68 infected group 4DPI (3/4), 7DPI (3/4), 14-28DPI (4/4) serum and lung (4/4) and spleen (4/4), detect viral nucleic acid with Real-time FQ-PCR method, and from serum, be separated to virus; And only from rV63 infected group 4DPI (2/4), 7DPI (3/4), 14DPI (3/4) 21-28DPI (4/4) serum and lung (3/4) and spleen (4/4), detect viral nucleic acid, from serum, be not separated to virus; Virus load in rV68 infected group serum and the tissue is higher than the rV63 infected group all the time, and wherein 14DPI, 21DPI and 28DPI serum have significant difference (P＜0.05), and lung tissue has utmost point significant difference (P＜0.01).IHC detects virus antigen and mainly is distributed in lung, thymus gland, spleen, tonsilla and hilar lymph node.The result shows, Nsp2 lacks viral rV63 the virulence of piglet is decreased, and has the potentiality that are developed further into marker vaccine.

1, material

(1) virus

RV68 and rV63 be through cultivating on the MARC-145 cell, the 13rd generation virus be used for the zoogenetic infection test.

(2) laboratory animal

The English of 21-25 age in days wean is that Large White is planted the pig farm available from Tianjin arm of the services portion, and continuous 5 years monitoring results show that this is the PRRS feminine gender, and test pig is all negative through serum antibody and viral nucleic acid detection PRRSV, PCV2." rainbow board 4075 " sucking pig mixed feed is available from Tianjin rainbow feed corporation,Ltd, and " mending gram doctor 8199 antibiotic breasts " 7-15kg piglet mixed feed in early stage is available from the inferior feed nutrition of Taiwan great achievement group Beijing Chinese Science and Technology Ltd..

(3) main agents

Porcine reproductive and respiratory syndrome virus antibody assay kit (PRRS 2XR) is available from American I DEXX company.HRP-conjugated AffiniPure Goat anti-mouse IgG (H+L) is available from Jackson ImmunoResearchLaboratories company.Lowlenthal serum, horse serum, poly-lysine, HRP mark goat anti-mouse IgG are available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing.DAB colouring reagents box is available from doctor's moral biotechnology company limited.

TRIzol ^LS available from Invitrogen company (Invitrogen life technologies, CA, USA); AMV ThermoScript II, Vsp I are available from Promega company.DNA glass milk fast purifying reclaims test kit available from the vast Imtech in Beijing.DTT, RNsin, dNTPs, 100bp DNA Ladder and Taq archaeal dna polymerase are Time Inc. available from sky, Beijing.SYBR ^Green Realtime PCR Master Mix is available from Japanese TOYOBO company.

DEPC, agarose are available from Sigma company.Other chemical reagent are homemade analytical pure product.

(4) primer

Table 4RT-PCR primer

The primer title	Primer sequence (5 ' → 3 ')	The position a	Goal gene	Clip size
					308F b	CAA ATA ACA ACG GCA AGC AG	14891～14910	N	308bp
308R b	AAA CTC CAC AGC GTA ACT TAT C	15177～15198	N
				P2327F c	ACC GCC TTT TCA CTG GCT AAC TAC	2327～2350	Nsp2	695bp(rV68) 632bp(rV63)
P2997R c	CCG CCG AAC TCA ATC TTT TCA CCT	2997～3020	Nsp2
				DetcM1 d	GAA CAA GTT TAA GGA GCT ACA GAC	8817～8841	Nsp9		793bp
DetcM2 d	ATT GGA CCT GAG TTT TTC CCA CAT	9586～9610	Nsp9

A: refer to position (the GenBank accession number: AF331831) in the BJ-4 genome; B: be used for negative animal screening of PRRSV and real-time fluorescence quantitative PCR; C: amplified fragments comprises the Nsp2 disappearance district of rV63; D: amplified fragments comprises the Vsp I genetic marker in clone's virus.

(5) key instrument equipment

DNA Engine Opticon ^2 Continuous Fluirescence Detector (quantitative real time PCR Instrument) are available from U.S. MJ Research company.

550microplate reader (microplate reader) is available from U.S. BIO-RAD company.

The rotary tissue processor of MICROM STP-120 is available from German MICROM company.

BMJ-1 biological tissue embedding machine is available from Tianjin Aviation Mechano-Electrical Co..

MICROM HM315 rotary microtome is available from German MICROM company.

PHY-III type pathological tissue floats the baking instrument available from the Zhongwei Electronic Instrument Factory, Zhangzhou City.

2, method and result

(1) experimentation on animals design, clinic observation and sampling

Animal grouping random packet, totally 3 groups (rV68 infected group, rV63 infected group, control group), 4 every group.Each organizes independent isolated rearing, prevents intersections such as personnel, feed, apparatus.

Feeding and management is kept the Animal House temperature about 20 ℃, and it is excessive to prevent that room temperature from changing.Give competent drinking-water of animal and feed, guarantee normal trophic level.

Infection will be cloned the MARC-145 cell culture of virus and normal MARC-145 cell multigelation 3 times, and 5000r/min is centrifugal, and 15min removes cell debris, collects supernatant, with MARC-145 raji cell assay Raji TCID ₅₀RV68 infected group and rV63 infected group via intranasal application respectively slowly splash into inoculation 5mL * 10 ^6.68TCID ₅₀/ mL (rV68) and 5mL * 10 ^7.00TCID ₅₀/ mL (rV63), each nostril 2.5mL, control group is with quadrat method inoculation 5mL MARC-145 cell conditioned medium.

Clinical observation is weighed in before connecing poison preceding (ODPI) and experiment end one by one, calculates and respectively organizes average daily gain.One by one measure rectal temperature during 14-16 afternoon every day, calculates and respectively organize mean body temperature every day, draws the body temperature change curve.Connect the clinical symptom of observing every pig day by day in detail behind the poison, mainly comprise situations such as spirit, quilt hair, skin, body condition, respiratory rate, cough, nasal secretion, appetite, movement.After the off-test, cut open all pigs extremely, observe pathology substantially one by one.

Sampling is carried out after infecting back 0,4,7,14,21,28 day (0DPI, 4DPI, 7DPI, 14DPI, 21DPI, 28DPI) and cuing open extremely.Wherein, DPI is the abbreviation of DAI (days post infection).

Through precaval vein blood sampling 5mL, aseptic separation of serum ,-80 ℃ of preservations are standby.

After the cotton swab of will sterilizing soaks in sterilization PBS (containing 2000U/mL penicillin and 2000 μ g/mL Streptomycin sulphates), go deep into every hog snout chamber postmedian sth. made by twisting and turn around (all carry out in the nostril, both sides), again cotton swab is placed 0.5mL sterilization PBS, standby in-80 ℃ of preservations.

When experiment finishes, cut open all pigs extremely, gather the heart, liver, spleen, lung, kidney, tonsilla, thymus gland, hilar lymph node, submandibular lymph nodes, inguinal lymph nodes, mesenteric lymph nodes, testis, ileum, fix with 4% Paraformaldehyde 96 immediately.And get lung tissue (right lung lobus diaphragmaticus end), the spleen tissue (end) of fixed position, standby in-80 ℃ of preservations separately.

(2) clinical symptom and cardinal principle pathology

By the beginning in the 4th day of intranasal inoculation clone virus back, sneezing appears in rV68 infected group and rV63 infected group pig successively, attaches most importance to 4-7 days, lasts till 14 days.Afterwards, indivedual pigs occur once in a while.After infection 4-11 days, a spot of serosity or mucus secretory product appearred in 4 of rV68 infected group and 4 hog snout chambeies of rV63 infected group.The rV68 infected group has 2 pigs (90309,90313) symptoms such as of short duration (3-5DPI) and slight lassitude, apocleisis, happiness be for sleeping in, inactive to occur.The rV68 infected group has 3 pigs (90309,90313,90601), rV63 infected group to have 1 pig (90407) laxativeness of a property crossed to occur respectively at 3-5DPI (infecting the back 3-5 days) and 6-7DPI (infecting the back 6-7 days).In addition, do not see that other are significantly unusual, body condition and the performance of other outward appearance and not obviously difference of control group.All are normal for control group.

Infect and cutd open inspection in back 32 days, the cardinal principle pathology (Figure 15) of interstitial pneumonia all appears in rV68 infected group and rV63 infected group pig.In addition, do not see other tangible pathology.Control group normal (Figure 15).Among Figure 15, A: control group; The B:rV63 infected group; The C:rV68 infected group.

(3) day weight gain

The body weight that increases according to every pig of experimental session is calculated the day weight gain of every pig divided by the experiment fate, and the average daily gain of rV68 infected group, rV63 infected group and control group is respectively (0.416 ± 0.054) kg, (0.414 ± 0.078) kg and (0.409 ± 0.068) kg as calculated.Carry out the significant difference analysis through the t check, infected group does not all have significant difference (P＞0.05) with control group, does not have significant difference (P＞0.05) between rV68 infected group and the rV63 infected group yet.

(4) body temperature changes

Infected back 7 days, the rV63 infected group has 1 temperature of pig body to be elevated to 40 ℃; Infected back 9 days, the rV68 infected group has 3 temperature of pig body to be elevated to (40 ℃, 40.2 ℃, 40.2 ℃) more than 40 ℃, though the slight trend of rising of infected group performance body temperature generally afterwards, with regard to individual speech instability also.Whole experimental session, the individual body temperature of infected group surpass 40 ℃ have respectively 18 times (rV63 infected group) and 17 inferior (rV68 infected group), and high fever all is 40.3 ℃; And the individual body temperature of control group surpasses 40 ℃ have only 3 times.Experimental session, each mean body temperature dynamic change (Figure 16) of organizing pig shows, from infecting back 9 days to 21 days, the mean body temperature of infected group is higher than control group always, and wherein the mean body temperature absolute value with 9-15DPI (infecting the back 9-15 days) (rV68 infected group), 9-16DPI (rV63 infected group) is a height.By above-mentioned data as can be known, can cause fervescence behind the two strain clone virus infection piglets, but not have notable difference between the infected group.Among Figure 16, Mock represents control group.

(5) Serum Antibody Detection

Carry out the ELISA antibody test according to porcine reproductive and respiratory syndrome virus antibody assay kit (PRRS 2XR) test kit specification sheets.The result shows that 2 infected group are all in 14DPI (infecting back 14 days), and 2/4 (in 4 two being arranged) pig antibody changes sun, infect 4 all commentaries on classics positive (4/4) in back 21 days, and control group keeps the ELISA negative antibody at whole experimental session always.The dynamic change of average S/P ratio such as Figure 17 of ELISA antibody.As seen from the figure, the antibody horizontal of infected group linearly rose later in infection in back 14 days, the average S/P ratio of each time point (poor/positive PRRS hole in S/P=sample P RRS hole and NHC hole and NHC hole poor) does not have significant difference (P＞0.05) though height is arranged mutually between t check demonstration rV68 infected group and rV63 infected group.Among Figure 17, Mock represents control group.

Employing fixed virus dilute serum method (Yin Zhen, Liu Jinghua. animal virology (second edition).Beijing: Science Press, 1997) carry out neutralization test with PRRSV BJ-4, all serum of collecting are detected, the result does not all detect neutralizing antibody.Use respectively with infected pigs homologous clone virus and carry out neutralization test, still do not detect neutralizing antibody.

(6) viremia

Utilize primer in the table 4 that the serum of (P2327F and P2997R) different time collection is carried out RT-PCR and detect, with 1.2% agarose gel electrophoresis inspection pcr amplification result.Result such as Figure 18 show the rV68 infected group in infection back 7 days, have 1 pig to begin to detect viral nucleic acid, infect back 14 days, infect that 1 and 3 pigs were arranged respectively in back 21 days is the viral nucleic acid positive (695bp), infect all to transfer feminine gender in back 28 days to; And the rV63 infected group has only a pig to detect viral nucleic acid (632bp) in back 21 days in back 14 days of infection, infection, infects to transfer feminine gender in back 28 days to.The viremia of rV68 infected group occurs 7 days than rV63 infected group is early, and the time length is longer than the latter, and virus detects positive rate also apparently higher than the latter.Among Figure 18, A:7DPI; B:14DPI; C:21DPI; 1: molecular weight standard; 2:rV68; 3:rV63; 4: negative control; The 5-8:rV68 infected group; The 9-12:rV63 infected group; 13-16: control group.

With the serum sample of aseptic collection MARC-145 cellular segregation virus, the result was separated to virus the serum in back 14 days from back 7 days of the infection of rV68 infected group, infection.The viral cultures that is separated to is carried out RT-PCR with primer DetcM1 and DetcM2, the purpose band of unique 793bp that increases.This purpose band can be cut into 540bp and 253bp two bands by Vsp I, and the amplified production of parental virus PRRSV BJ-4 can not be cut (Figure 19) by Vsp I.Among Figure 19,1: molecular weight standard; 2:BJ-4 RT-PCR amplified production is cut the result through Vsp I enzyme; 3-4: isolated viral RT-PCR amplified production is cut the result through Vsp I enzyme.

Carry out sequencing behind the RT-PCR product purification to isolated viral, result (Figure 20) confirms that Vsp I still exists.Above-mentioned test-results shows that clone's virus is duplicated in the pig body after, the artificial genetic marker of introducing heredity stably in its genome.Two parts of serum samples that are separated to virus are carried out TCID ₅₀Measure, rV68 and rV63 poison valency is respectively 10 as a result ^4.500TCID ₅₀/ mL and 10 ^3.667TCID ₅₀/ mL.

(7) virus load in serum and the tissue

In order further the virus load in viremia and the tissue to be carried out quantitative analysis, lung after serum that different time is collected and animal experiment finish and spleen tissue carry out real-time fluorescence quantitative PCR and detect.Concrete grammar is as follows:

With the TE damping fluid with standard plasmid-PRRSV recombinant plasmid (execute and start. porcine reproductive and respiratory syndrome virus and porcine circovirus 2 type coinfection are to the cell and the molecular mechanism of pig Immune Function: [doctorate paper].Beijing: China Agricultural University, 2006) carry out 10 times and increase progressively dilution continuously, get 10 ^-2-10 ^-8(promptly 6.19 * 10 ¹⁰-6.19 * 10 ⁴Copies/ μ L) as template, so that the production standard curve.

Taking-up is set up 20 μ L reaction systems with cDNA 4 μ L (serum), 2 μ L (tissue) and the standard plasmid 1 μ L of oligo (dT) 18 reverse transcriptions, and each sample repeats twice.

SYBR ^Green Realtime PCR Master Mix 10μL

308F(25pmol/μL) 0.5μL

308R(25pmol/μL) 0.5μL

Template 1-4 μ L

H ₂O 8-5μL

Real-time FQ-PCR loop parameter is: 95 ℃ of 5min, and 94 ℃ of 20s, 55 ℃ of 20s, 72 ℃ of 30s read plate 1s for 84.8 ℃, after 40 circulations, 72 ℃ of 5min.At last, between 65 ℃ ~ 95 ℃, read plate 1 time for per 0.2 ℃, each 1s.

With software MJOpticon monitor ^TMAnalysis software Ver 3.1 analyzes and calculation result.At last, get the amplified production electrophoresis, the specificity of checking amplification.

Result (Figure 21 A) by standard plasmid has obtained typical curve (Figure 21 B), has set up linear regression equation (y=-0.2881x+13.99), its relation conefficient (r ²) reach 0.995.Single fusion peak (Figure 21 C) and single amplified production (Figure 21 D) show that detected result has very strong specificity.Among Figure 21, A: data plot; B: typical curve; C: melting curve; D: agarose gel electrophoresis.

Sample detection result's (table 5) shows that two infected group can detect viral nucleic acid from the later serum of 4DPI and lung and spleen tissue, but the recall rate of the serum of rV63 infected group 4DPI, 7DPI, 14DPI and lung tissue is lower than rV68 infected group; And in rV68 infected group 14DPI, 21DPI, 28DPI serum and the lung tissue virus carrying capacity significantly or the utmost point be significantly higher than the rV63 infected group.All do not detect virus in the serum of control group and the tissue.

Viral nucleic acid positive rate and carrying capacity (Log thereof in table 5 serum and the tissue ₁₀Copies/ml or Log ₁₀Copies/g)

Sample	The rV68 infected group		The rV63 infected group		The P value	Difference
							Recall rate	Detected level	Recall rate	Detected level
	Serum (4DPI)	3/4	3.302±2.104	2/4							1.740±1.917	0.143	Not remarkable
Serum (7DPI)					3/4	4.108±1.975	3/4	3.111±1.988	0.227	Not remarkable
	Serum (14DPI)	4/4	5.212±0.663	3/4							3.289±2.124	0.028	Significantly
Serum (21DPI)					4/4	5.680±0.623	4/4	4.976±0.470	0.024	Significantly
	Serum (28DPI)	4/4	5.164±0.261	4/4							4.896±0.176	0.031	Significantly
Lungs					4/4	7.625±1.945	3/4	3.894±2.442	0.004	Extremely remarkable
	Spleen
		4/4	5.671±0.361	4/4	5.317±0.601	0.175	Not remarkable

(8) nasal cavity toxin expelling

All nasal cavity swabs of gathering are carried out RT-PCR, all do not detect viral nucleic acid.Nose swab washing lotion supernatant continuous 5 generations of blind passage on the MARC-145 cell, be not separated to virus.

(9) virus in the tissue distributes

After off-test, slaughter pig, getting the heart, liver, spleen, lung, kidney, tonsilla, thymus gland, submandibular lymph nodes, hilar lymph node, mesenteric lymph nodes, inguinal lymph nodes, ileum, the testis tissue of every pig, is that an anti-immunohistochemical methods that carries out detects the virus antigen distribution with N albumen McAb SDOW17.The result detects virus antigen at lung, hilar lymph node, thymus gland, spleen, tonsilla, the inguinal lymph nodes of test group, and control group organize with other of test group in a organized way and all do not detect virus antigen (Figure 22).Among Figure 22, A-F: infected group; G-L: control group; A, G: lung; B, H: hilar lymph node; C, I: thymus gland: D, J: spleen; E, K: tonsilla; F, L: inguinal lymph nodes (infected back 32 days, 200 *)

Virus antigen between two infected group distributes and the recall rate of various tissues does not have notable difference (table 6).

The distribution and the positive ratio of virus antigen in the table 6 infected pigs tissue

Tissue	Lung	Spleen	Thymus gland	Tonsilla	Hilar lymph node	Inguinal lymph nodes
							rV63
	3/4	2/4	3/4	1/4	4/4	1/4
							rV68	4/4	2/4	2/4	2/4	4/4	1/4

(10) the main difference of rV68 infected group and rV63 infected group

Every detection index to rV68 infected group and rV63 infected group is added up, and main difference sees Table 7.

The main difference of table 7rV68 infected group and rV63 infected group

Group	Clinical manifestation			Viremia (conventional PCR)		Virus load (quantitative fluorescent PCR)		Serum-virus separates
									Nasal discharge	Depressed apocleisis	Diarrhoea	Time length	Supreme Procuratorate goes out rate	Recall rate	Detected level
	rV68
		2/4	2/4	3/4	7～21DPI	3/4(21DPI)	3/4～4/4	RV68 group serum (14,21DPI, 28DPI) and lung tissue are significantly higher than rV63 and organize	2/4
	rV63									3/4	0/4	1/4	14～21DPI	1/4(14、21DPI)	2/4～4/4	0/4

Sum up

Ideal infections clone virus should keep and parental virus in full accord in heredity, but in fact this is the thing that is difficult to accomplish.Be not only because " quasispecies " feature (Goldberg et al, 2003 of PRRSV itself; Rowlandet al.et al, 1999), 5 ' end base and the 3 ' Poly (A) that be difficult to accurately measure, also have fidelity problem (Bracho et al, 1998 of archaeal dna polymerase and phage rna polymerase; Cline et al, 1996).In fact in the infections clone process that makes up BJ-4 and other strain, all finds jumping phenomenon, even caused biological characteristics generation variation (Nielsen et al, 2003 to a certain degree of clone's virus of having; Truong et al, 2004).Therefore, the biological characteristics of thoroughly evaluating clone virus becomes the important step of research work.The viral rV68 that comes from the wild-type infectious CDNA clones is with heavy dose of (the slight upper respiratory tract infection of performance behind 5 * 106.667TCID50) the infection weanling pigs, slight of short duration diarrhoea, the depressed and apocleisis of spirit, symptoms such as the slight rising of body temperature; Pathology mainly is an interstitial pneumonia substantially; The virus antigen limitation is distributed in lung, spleen, thymus gland, tonsilla and indivedual lymphoglandula; Detect with conventional RT-PCR, viremia comes across 7DPI first, the 28DPI completely dissolve, and from the serum of two pig, be separated to virus, but the virus titer in the serum is lower; 14DPI produces ELISA antibody, and continues to raise in duration of test average antibody level; Average daily gain is not subjected to obviously to influence; Do not detect the nasal cavity toxin expelling yet.With parental virus BJ-4 infect piglet the result (influence and viral nucleic acid distribution dynamic that Zheng Jie .PRRS virus persistent infection pair cell factor R NA transcribes: [master thesis]. Beijing: China Agricultural University, 2002) compare, the two is at aspect no significant differences such as body temperature, respiratory symptoms, but the viremia obvious difference.The piglet that BJ-4 infects all detects viral nucleic acid from the serum of 1DPI, 2DPI, 4DPI, 7DPI, last till 21DPI always; And after the rV68 infection, just have the serum of (a 1/4) pig viral nucleic acid to occur up to 7DPI.In addition, BJ-4 inguinal lymph nodes, iliac lymph nodes, shoulder ALN and the tangible enlargement of mesenteric lymph nodes occur after infecting, and is that rV68 infects the cardinal principle pathology that is not had.And the infective dose of rV68 is that the wild-type that shows more than 10 times of BJ-4 is cloned viral rV68 pathogenic lower than parental virus BJ-4 to piglet in this test.Illustrate that wild-type clones viral rV68 pathogenic lower than parental virus BJ-4 to piglet.This species diversity occurring may be that 23 point mutation that exist in clone's viral genome cause.Because in making up the infections clone process, these 23 point mutation are not replied, wherein 12 are caused amino-acid substitution, may influence virus replication in vivo, and part key amino acid itself and virus virulence that also may be wherein be closely related.A kind of possibility is high wherein, and the breeding ratio parent's poison BJ-4 on the MARC-145 cell is slow slightly because the test of front shows rV68, and the result is more identical with in vivo test.The clone's virus that has in vivo, external test-results and inconsistent, as american type prototype strain VR-2332, although its clone's virus is lower than parent's poison in external growth titre, but still keep the highly pathogenicity (Nielsen et al, 2003) of parental virus to susceptible animal.This may be that the environment that more helps virus multiplication in the natural reservoir (of bird flu viruses) body has overcome some and suppresses the factor of the viral in-vitro multiplication of clone, makes it keep highly pathogenicity.In addition, do not get rid of that some unfavorable sudden changes are duplicated in vivo with virus and the possibility that is able to reverse mutation yet.

21 amino acid whose rV63 are with similar dosage (5 * 10 for the Nsp2 disappearance ^7.000TCID ₅₀) infect piglet after, except not seeing the depressed and apocleisis of tangible spirit, in clinical symptom, pathology, serum antibody dynamic change, virus antigen distribute substantially, aspect such as nasal cavity toxin expelling and day weight gain and rV68 infected group do not have notable difference.But, the obvious difference of viremia.Detect with conventional RT-PCR, the rV63 infected group has only a pig to detect viral nucleic acid when 14DPI, 21DPI, and 28DPI disappears.The positive rate of viremia and time length are starkly lower than or are shorter than the rV68 infected group.Detect with real-time fluorescence quantitative PCR, the serum of rV68 infected group 4DPI, 7DPI, 14DPI and the virus-positive rate in the lung tissue are higher than the rV63 infected group, and the serum of the former 14DPI, 21DPI, 28DPI and the virus load in the lung tissue significantly or the utmost point be significantly higher than the latter.During the whole test, be not separated to virus yet, and from the serum of rV68 infected group, be separated to two strain virus from the rV63 infected group.By The above results as can be known, rV63's is pathogenic lower than rV68.As if yet in vitro tests shows that rV63 has the growth characteristics stronger than rV68 on the MARC-145 cell, and is opposite with experimental result in the body.In fact, this also is understood that.At first, the microenvironment in the passage cell and the body environment of natural reservoir (of bird flu viruses) have very big difference.Secondly, the adaptive faculty of virus on cell can not be represented its virulence to host animal.With regard to PRRSV, the evidence of two aspects is arranged.The one, still be that the adaptability of passage cell all presents certain otherness to PAM between the strain isolated of different virulence, particularly some virulent strains can not adapt to passage cell and cultivate (Bautista et al, 1993; Mengeling et al, 1995); On the other hand, the attenuated vaccine that has is to the adaptability of passage cell apparent altitude, and pig only demonstrated (Allende etal, 2000 of weakening of virulence; Yuan et al, 2001), even the strain that has report passage cell height to adapt to has been lost the infectivity (B  tner et al, 1999) to natural target cell PAM.

After experiment showed PRRSV infected pigs, neutralizing antibody produced than later, and the time of bibliographical information is also inconsistent, can just detect early at 8DPI, will arrive 56DPI and just can detect, detection time first (Batista et al, 2004 between 14-35DPI that great majority are tested of evening; Diaz et al, 2005; Labarque et al, 2000; Takikawa etal, 1997; Yoon et al, 1995).In this research, on the MARC-145 cell, measures the serum neutralizing antibody of cloning viral infected pigs as indicator virus, all do not detect, use rV68, rV63 instead and heavily examine still and do not detect until 28DPI with parental virus PRRSV BJ-4.But do not detect neutralizing antibody, do not show not produce neutralizing antibody in the animal body.Because be not have neutralizing antibody to produce in the serum of up-to-date result of study demonstration infection animal, but the susceptibility of the serious passivation neutralization test of N glycosylation on the indicator virus GP5, the also generation (Ansari et al, 2006) of the interior neutralizing antibody of severe inhibition body of the glycosylation of GP5 in addition.And have 4 N glycosylation sites at PPRSV BJ-4 and near cloning the neutralizing epitope of viral GP5.Not detecting neutralizing antibody may be relevant therewith.

From rV68 infected group serum sample, be separated to virus, cut through RT-PCR and enzyme and identify and sequential analysis, show that the genetic marker of introducing still exists, show that clone's virus is stable in vivo to duplicate.

Brief summary

The heavy dose of infection of the crohn poisons nasal piglet of A, two strains rescue does not cause that tangible clinical disease takes place, and main performance is sneezed, clinical symptom such as the slight rising of body temperature.Cut open the inspection pathology and only limit to interstitial pneumonia, the viremia time length is short, overall positive rate is low, do not detect the nasal cavity toxin expelling, virus antigen mainly is distributed in lung, spleen, thymus gland, tonsilla and minority lymphoglandula, average daily gain and control group do not have significant difference, show viral rV68 of clone and the rV63 pathogenic reduction to piglet.

B, detect with conventional RT-PCR, the rV63 infected group has only 1 pig to detect viral nucleic acid at 14DPI and 21DPI, and the rV68 infected group has 3 pigs viremia to occur, and wherein 1 lasts till 21DPI from 7DPI; Detect with real-time FQ-PCR, viral level in rV68 infected group serum and the tissue is higher than the rV63 infected group, and the content in 14DPI, 21DPI, 28DPI serum and the lungs has significantly or utmost point significant difference, the virulence that shows the rV63 of Nsp2 excalation further reduces, and can be used as candidate's strain of exploitation marker vaccine.

C, rV63 and rV68 infected group all produce ELISA antibody in 14DPI, and all change sun during 21DPI, but still do not detect neutralizing antibody until 28DPI, might be to be subjected to the glycosylated interference of viral protein.

D, be separated to virus from rV68 infected group serum sample, the intact reservation of its genetic marker shows that clone's virus duplicates and have inheritance stability in the pig body

In a word, zoogenetic infection test shows that rV68's is pathogenic lower than parental virus, and the virulence of rV63 has further and weakens.Because the Nsp2 of rV63 lacks 21 amino acid, have the potentiality that are developed to marker vaccine, and the polypeptide that can lack exploitation differential diagnosis method.In exploitation vaccine process, can utilize the reverse genetic technology platform of having set up that the glycosylation site of GP5 is transformed, overcome the damper that influences neutralizing epitope, to improve vaccine-induced neutralizing antibody level.

Claims (5)

1. method that reduces PRRSV BJ-4 strain virulence is that length is the nucleotide fragments of 69nt in the Nsp2 coding region of consecutive miss PRRSV BJ-4 pnca gene group, obtains the strain that virulence reduces; The nucleotide sequence of described 69nt nucleotide fragments as GenBank Accession Number AF331831 from 5 ' terminal the 2795th to 2863 shown in.

2. method according to claim 1, it is characterized in that: in the described method, length is on the basis of nucleotide fragments of 69nt in the Nsp2 coding region of described consecutive miss PRRSVBJ-4 strain, inserts restriction endonuclease sites at GenBank AccessionNumber AF331831 in 5 ' terminal the 2795th to 2863 again.

3. method according to claim 2 is characterized in that: described restriction enzyme is HpaI.

4. the strain that obtains by arbitrary described method in the claim 1 to 3.

5. the described strain of claim 4 prevents and/or treats application in the porcine reproductive and respiratory syndrome vaccine in preparation.

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