CN114317574A

CN114317574A - Cultivation and application of Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome virus

Info

Publication number: CN114317574A
Application number: CN202111619805.0A
Authority: CN
Inventors: 陈南华; 李树滨; 朱建中
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2022-04-12
Anticipated expiration: 2041-12-28
Also published as: CN114317574B

Abstract

The invention relates to cultivation and application of a passage Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome modified virus, belonging to the technical field of bioengineering. The invention provides a PRRSV1 modified virus which can adapt to Marc-145 cell in-vitro subculture. Based on an HLJB1 infectious cloning platform which cannot adapt to the PRRSV1 wild strain of the Marc-145 cell, the rescued modified virus is constructed by continuously modifying ORF2 and ORF3 genes, and can adapt to the in-vitro continuous subculture of the Marc-145 cell. Meanwhile, the modified virus is inoculated to pig bodies without causing morbidity and can induce the generation of a neutralizing antibody of PRRSV1, so that the modified virus can be used as a PRRSV1 live vector vaccine candidate strain for developing a novel genetic engineering vaccine for resisting PRRSV1 epidemic strains in China. The successful development of the PRRSV1 specific genetic engineering live vaccine has great economic value and good market prospect.

Description

Cultivation and application of Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome virus

Technical Field

The invention relates to a Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome modified virus cultured by a reverse genetic manipulation technology and application thereof, belonging to the technical field of bioengineering.

Background

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) seriously compromises the healthy development of the global swine industry. PRRSV infects all day-old pigs and is primarily characterized by abortions in sows and respiratory syndromes in piglets. PRRSV strains can be classified as: PRRSV1 and PRRSV 2. The main prevalence in China is PRRSV2, but in recent years PRRSV1 has also existed widely in China and has a tendency to further prevalence. In addition, in Sichuan, Guangdong, etc., an epidemic has been reported to be caused by PRRSV1 infection.

PRRSV belongs to the family arterivirus and is a single-stranded positive-strand RNA virus with a genome size of about 15kb containing 10 Open Reading Frames (ORFs). ORF1a and ORF1b encode at least 16 non-structural proteins, and ORF2-7 encodes 8 structural proteins. Among them, three small envelope proteins (GP2a, GP3 and GP4) encoded by ORF2-4 gene form heterotrimers through non-covalent bonds and play a key role in infecting target cells with PRRSV. Research shows that the adaptability of PRRSV1 strain to Marc-145 cells can be obviously improved by mutating amino acid 88/94/95 of GP2 a.

Vaccine immunization is an effective means for controlling PRRS epidemic. Although PRRSV1 has become widely prevalent in our country in recent years, no PRRSV1 specific vaccine has been developed in our country. Because the traditional PRRSV1 wild strain in China can only be proliferated on primary alveolar macrophage PAM and cannot be cultured on passage Marc-145 cells. This is a great problem in developing PRRSV1 live vaccine.

Reverse genetic manipulation has become an important means for the development of novel genetic engineering vaccines. However, no report is available for obtaining the PRRSV1 wild strain which can adapt to Marc-145 cells by using reverse genetic manipulation technology.

Disclosure of Invention

Aiming at the problem that the current China lacks a commercial vaccine for effectively preventing and controlling the PRRSV1 epidemic strain, the invention provides a PRRSV1 modified strain which is suitable for Marc-145 cell in-vitro subculture. The PRRSV1 modified strain can be used as a candidate strain for developing PRRSV1 genetic engineering live vaccine. In particular to the cultivation and the application of Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome virus.

The invention is realized by the following technical scheme:

a recombinant plasmid is used for preparing Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome modified virus, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 1.

The Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome modified virus rHLJB1-M-Ame3 is rescued and prepared by the recombinant plasmid.

The Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome modified virus rHLJB1-M-Ame3 construction method comprises the following steps:

the method comprises the steps of firstly constructing infectious clone virus rHLJB1 for rescuing the PRRSV1 wild strain which cannot adapt to the Marc-145 cells, then carrying out site-specific mutation on the ORF2 of the vesicular membrane protein GP2a encoding gene, subsequently replacing the ORF3 of the vesicular membrane protein GP3 encoding gene by homologous recombination, and finally successfully rescuing the PRRSV1 modified strain rHLJB1-M-Ame3 which can adapt to the Marc-145 cells through the double modification of the ORF2 and the ORF3 genes.

The invention provides a PRRSV1 strain rHLJB1-M-Ame3 which is modified and cultured by a reverse genetic manipulation technology and can adapt to the subculture of Marc-145 cells. The parent strain HLJB1 can only adapt to PAM primary cell in vitro culture before modification, and the modified rHLJB1-M-Ame3 can adapt to Marc-145 subculture cells. The modified virus greatly facilitates later application in live vaccine development.

Further, the infectious clone virus rHLJB1 of the PRRSV1 wild strain is: five single restriction sites SgsI, Pfl23II, BglII, Bsp1407I and XbaI are inserted into the pACYC177 vector by using a pACYC177 low-copy vector through gene synthesis and homologous recombination, and are used for constructing infectious clone virus rHLJB 1; the rHLJB1 contains a genome full-length cDNA sequence of PRRSV1 wild strain HLJB1, the 5 'end of the full-length cDNA sequence is added with a cytomegalovirus eukaryotic promoter sequence, the 3' end Poly (A) tail downstream is added with a hepatitis D virus ribozyme sequence and a bovine growth hormone polyadenylation signal transcription termination sequence.

Further, the construction method of the Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome modified virus rHLJB1-M-Ame3 comprises the following steps:

1) synthesizing pUC57-Synthesis-Amervac-ORF2-4 plasmid;

2) designing a primer;

3) construction of an infectious clone of HLJB1-M-Ame 3;

3-1) HLJB1-M-Ame3 whole gene segmented amplification;

3-2) connecting the HLJB1-M-Ame3 fragments;

4) rescue of infectious clonal virus of rHLJB1-M-Ame 3.

Further, the primers designed in step 2) are as follows:

further, the Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome modified virus rHLJB1-M-Ame3 is applied to preparation of novel genetic engineering vaccines.

On the other hand, the invention provides an artificially modified PRRSV1 genetic engineering live vaccine candidate strain, and the artificially modified PRRSV1 modified strain which is suitable for Marc-145 cells and is obtained by the artificial modification can not cause the temperature rise, does not influence the weight gain, does not cause the morbidity and mortality of piglets after being inoculated with the piglets, and has good safety. Meanwhile, the PRRSV1 specific neutralizing antibody can be induced, so that the PRRSV1 specific neutralizing antibody can be used as a genetic engineering vaccine candidate strain to be applied to culture a PRRSV1 genetic engineering live vaccine.

In another aspect of the invention, the artificially modified PRRSV1 strain is applied to research of a Marc-45 cell adaptability mechanism. Specifically, a reverse genetic operation technology is utilized to continuously carry out site-directed mutation and fragment replacement on the saccule membrane protein coding gene of the PRRSV1 modified strain which is suitable for the Marc-145 cell and the PRRSV1 wild strain which is not suitable for the Marc-145 cell, so that the key gene and site for determining the adaptability of the Marc-145 cell of the PRRSV1 wild strain are accurately analyzed.

Compared with the prior art, the invention has the following beneficial technical effects:

1. the modified infectious clone virus is a PRRSV1 modified strain which can adapt to Marc-145 cell in-vitro subculture;

2. the constructed modified virus has good in vivo and in vitro proliferation efficacy, cytopathic effect (CPE) can be caused by infecting Marc-145 passage cells, and specific viremia can occur when a pig body is inoculated;

3. the constructed modified virus has good safety, does not cause fever after being inoculated to a pig body (the body temperature is less than 40 ℃), does not influence weight gain, and does not cause death of piglets;

4. the constructed modified virus can induce protective immune response, and the inoculation pig can induce and generate a PRRSV1 neutralizing antibody;

5. the constructed modified virus can be used for developing PRRSV1 gene engineering live vaccines, and is beneficial to the prevention and control of PRRS epidemic situation in China.

Drawings

FIG. 1 is a schematic diagram of the construction of the full-length cDNA ligation of rHLJB1-M-Ame3 modified virus genome in example 1 of the present invention;

FIG. 2 is a diagram of the cellular pathology caused by infection of Marc-145 cells with rHLJB1-M-Ame3 modified virus in example 1 of the present invention;

FIG. 3 is a graph of the results of detection of rescued rHLJB1-M-Ame3 engineered virus in Marc-145 cells using an indirect immunofluorescence assay against a monoclonal antibody to the N protein of PRRSV in example 1 of the present invention;

FIG. 4 is a graph of the results of detection of rescued rHLJB1-M-Ame3 engineered viruses in PAM cells using an indirect immunofluorescence assay against a monoclonal antibody to the N protein of PRRSV in example 1 of the present invention;

FIG. 5 is a graph showing the results of detecting the dynamic proliferation of rHLJB1-M-Ame3 modified virus on Marc-145 cells by using a fluorescent quantitative PCR method in example 1 of the present invention;

FIG. 6 is a graph showing plaque results of Marc-145 cells infected with the rHLJB1-M-Ame3 modified virus in example 1 of the present invention;

FIG. 7 is a graph showing the results of viremia induced by rHLJB1-M-Ame3 modified virus inoculated swine in example 2 of the present invention;

FIG. 8 is a graph showing the results of body temperature changes of pigs inoculated with rHLJB1-M-Ame3 modified virus in example 2 of the present invention;

FIG. 9 is a graph showing the results of body weight changes of pigs inoculated with rHLJB1-M-Ame3 modified virus in example 2 of the present invention.

Description of the nucleotide sequence

The SEQ ID NO.1 recombinant plasmid rHLJB1-M-Ame3 sequence;

SEQ ID NO.2 primer HLJB 1-ASCI-F1;

SEQ ID NO.3 primer HLJB1-Pfl23 II-R1;

SEQ ID NO.4 primer HLJB1-Pfl23 II-F2;

SEQ ID NO.5 primer HLJB 1-BglII-R2;

SEQ ID NO.6 primer HLJB 1-BglII-F3;

SEQ ID NO.7 primer HLJB1-Bsp 1407I-R3;

SEQ ID NO.8 primer HLJB1-Bsp 1407I-F4;

SEQ ID NO.9 primer HLJB 1-R4-1;

SEQ ID NO.10 primer HLJB 1-XbaI-R4-2;

SEQ ID NO.11 primer HLJB1-ORF2 aM-F;

primer HLJB1-ORF2aM-R of SEQ ID NO. 12;

primer Amervac-overlap-ORF2a-R of SEQ ID NO. 13;

primer Amervac-overlap-ORF3-F of SEQ ID NO. 14;

Detailed Description

For routine experimentation in the following examples, see molecular cloning, A laboratory Manual, third edition, by Sambrook et al (Beijing: scientific Press, 2002), the use of the instrument is described with reference to instructions for the operation of the instrument.

In the examples of the present invention, the virus has an isolate of HLJB 1. The cells include BHK-21 cell line and primary alveolar macrophage PAM and Marc-145 cells.

In the examples of the present invention, plasmids and strains: pACYC177 plasmid was purchased from Youbao organisms, rHLJB1 infectious clone plasmid was stored in the laboratory (construction methods described above), and Trans1-T1 competent cells were purchased from Beijing Quanjin organisms, Inc., pUC57-Synthesis-Amervac-ORF2-4 and pACYC177-New were synthesized by Kinzhi, Suzhou.

In the examples of the present invention, other reagents used: RNase Free H2O, pancreatin cell digest (phenol red) from Solambio; the TRIpure Reagent for total RNA extraction was purchased from Eldely Bio Inc; PrimeScript1st Strand cDNA Synthesis Kit, 2 XPrimeSTAR MAX DNA Polymearse from TAKARA; FastPure Plasmid Mini Kit was purchased from Novovozan Biotech Co., Ltd; DMEM media was purchased from HyClone biochemicals ltd; fetal bovine serum was purchased from Sigma; DyLight 594, Goat anti-Mouse IgG (H + L) Secondary Antibody, purchased from Invitrogen; DNA Marker was purchased from Zhejiang Bo and jin science and technology Co., Ltd; 2 × Biogold Tag Plus PCR MasterMix was purchased from Zhejiang Bo and gold science, Inc.; homologous recombination Kit Cloneexpress MultiS One Step Cloning Kit was purchased from Vazyme; gel Extraction Kit was purchased from Beijing kang, a century Biotechnology Co., Ltd; DNA restriction enzymes were purchased from Thermo Fisher; t4 DNA Ligase, Lipofectamine^TM3000Transfection Reagent was purchased from Invitrogen.

Example 1

The transformation, construction and rescue of the type 1 porcine reproductive and respiratory syndrome virus which can adapt to the in vitro culture of Marc-145 cells;

1.1 plasmid Synthesis of pUC57-Synthesis-Amervac-ORF2-4

According to the complete gene sequence (GenBank accession number: GU067771.1) of PRRSV1 strain Amervac adaptable to Marc-145 cells in GenBank, the ORF2-4 gene sequence is artificially synthesized by Jinwei corporation, and the total length is 1702 bp. Obtaining pUC57-Synthesis-Amervac-ORF2-4 plasmid.

1.2 primer design

Amplification primers containing the ORF3 gene of Amervac were designed using Primer 5.0, with upstream and downstream primers containing homology arms matching the ORF2 and ORF4 genes of HLJB1, respectively, for the construction of the chimeric F3 fragment by overlap PCR as shown in fig. 1.

Designing a mutation primer, and introducing 3 point mutations into the ORF2 gene of the HLJB1 to change the amino acids at the 88 th, 94 th and 95 th positions from IMF to FIL.

The 4 primers comprising the amplification of the entire gene of HLJB1 were designed in the previous construction of PRRSV1 infectious clone rHLJB1, and all primers used are detailed in table 1 below:

TABLE 1 primers for construction of PRRSV infectious clone engineered viruses of the present invention

1.3 construction of an infectious clone of HLJB1-M-Ame3

1.3.1HLJB1-M-Ame3 Whole Gene segment amplification

As shown in FIG. 1, HLJB1-M-Ame3 has a total of 4 segments. Firstly, HLJB1 virus is taken as a template, and HLJB1-SgsI-F1 and HLJB1-Pfl23II-R1 are respectively taken; HLJB1-Pfl23II-F2 and HLJB1-BglII-R2 primer pair combination; f1 and F2 fragments were amplified. Secondly, the F4 fragment needs to be amplified in two steps, namely, firstly, the F4-1 fragment is amplified by using HLJB1-Bsp1407I-F4 and HLJB1-1R4 in a pairing mode, then the F4-1 amplification product is used as a template, and the F4 fragment is obtained by using HLJB1-Bsp1407I-F4 and HLJB1-XbaI-2R4 primer pair in an amplification mode, and the hepatitis D virus (HDV Ribozyme) sequence is added.

The F3 fragment amplification of HLJB1-M-Ame3 needs to be carried out in multiple steps, firstly, HLJB1 virus is taken as a template, and HLJB1-BglII-F3 and HLJB1-ORF2aM-R are respectively taken; HLJB1-ORF2aM-F and HLJB1-Bsp1407I-R3 primer pairs are amplified to obtain F3-M1 and F3-M2. Then, F3-M1 and F3-M2 are used as templates, and HLJB1-BglII-F3 and HLJB1-Bsp1407I-R3 primer pairs are used for carrying out overlap PCR to obtain F3-M3 with 88/94/95 three amino acid mutations. And amplifying by using an HLJB1-BglII-F3 primer pair and an Amervac-overlap-ORF2a-R primer pair by using F3-M3 as a template to obtain F3-Ame 3-1. Then, using pUC 57-Synthesis-America-ORF 2-4 synthetic plasmid as a template, and using America-overlap-ORF 3-F and HLJB1-Bsp1407I-R3 primer pair to amplify to obtain F3-Ame 3-2. And finally, performing overlap PCR by using F3-Ame3-1 and F3-Ame3-2 as templates and HLJB1-BglII-F3 and HLJB1-Bsp1407I-R3 primer pairs to obtain F3-Ame3-3 through amplification. The reaction system and the reaction program of HLJB1 segmented amplification are specifically as follows:

TABLE 2 reaction System

cDNA	2×PrimeSTAR MAX DNA Polymearse	Primer (10. mu.M)	H₂O
				2μL	20μL	1μL	Make up to 40 mu L

TABLE 3 reaction procedure

F1, F2 and F4 fragments of HLJB1, and F3-Ame3-3 fragment with three amino acid mutations and Amervac ORF3 gene were amplified separately according to the above method. Taking 2 mu L of PCR reaction product, and carrying out electrophoresis by using agarose gel with the concentration of 0.9% to respectively obtain four gene fragments with the fragment sizes of 4371bp, 5230bp, 2001bp and 3758 bp.

1.3.2 connection of each fragment of HLJB1-M-Ame 3;

the amplification product was purified using a product purification kit. The purified product is subjected to double enzyme digestion with pACYC177-New plasmid, and is sequentially connected to pACYC177-New vector, and the construction strategy is shown in figure 1. The specific operation steps are as follows:

firstly, carrying out double digestion on PACYC177-New plasmid and HLJB 1F 1 amplification products by using Sgs I and Pf l23II, wherein the PACYC177-New enzyme digestion system is as follows: 15 μ L of PACYC177-New plasmid (200 ng/. mu.L), SgsI3 μ L, Pfl23II 3 μ L,10 × CutSmart Buffer 4 μ L, RNase Free H₂And O is supplemented to 40 mu L. The enzyme cutting system of HLJB 1F 1 is as follows: purified product of HLJB 1F 1 (200 ng/. mu.L) 10. mu.L, SgsI 2. mu.L, Pfl23II 2. mu.L, 10 × CutSmart Buffer 4. mu.L, RNase Free H₂And O is supplemented to 40 mu L. Reaction conditions are as follows: water bath at 37 ℃ for 30 minutes.

Separating the enzyme-digested band by agarose gel electrophoresis, cutting the target band and carrying out gel recovery. The linear vector pACYC177-New recovered from the gel and the enzyme digestion product HLJB 1F 1 are connected by T4 DNA ligase according to the molar ratio of 1:8, and then Trans1-T1 competent cells are transformed, independent colonies are picked for pure culture, and detection primers are used for carrying out PCR detection on bacteria liquid. Selecting PCR positive bacteria for overnight enrichment culture, extracting plasmid DNA, carrying out double enzyme digestion identification by using SgsI and Pfl23II, carrying out 0.8% agarose gel electrophoresis, selecting the plasmid with the correct enzyme digestion size, carrying out sequencing verification, and then continuing to carry out next fragment ligation. The F2, F3-Ame3-3 and F4 fragments are connected by a similar method to the F1 connection method according to the construction strategy diagram shown in FIG. 1, and finally the infectious clone plasmid rHLJB1-M-Ame3 containing the whole genome sequence is obtained. The full-length sequence of the rHLJB1-M-Ame3 recombinant plasmid is detailed in SEQ ID NO. 1. When the F3-M3 fragment containing only three-point ORF2 icons is used to replace F3-Ame3-3, the infectious clone plasmid rHLJB1-M can be constructed.

1.4 rescue of infectious clonal virus of rHLJB1-M-Ame 3;

BHK-21 cells were previously cultured at 5X 10 in DMEM medium containing 10% FBS⁵cells/well density were plated in 12-well cell culture plates at 37 ℃ in 5% CO₂The culture box is used for culturing until the cell density reaches about 90 percent. According to Lipofectamine^TM3000Transfection Reagent indicated cell Transfection, the procedure was as follows:

first, plasmid premix was prepared in an EP tube: 2 μ g of rHLJB1-M-Ame3 infectious clone plasmid, P30004 μ L, and Optin-MEM 50 μ L; in another EP tube a Lip3000 premix was placed: lip30003 μ L, Optin-MEM 50 μ L; and finally, mixing the premixed solution obtained in the two steps, standing at room temperature for 15min, and adding cells to be transfected. After 36-48h of cell transfection, the supernatant was collected and frozen at-80 ℃ for use.

Marc-145 cells were cultured at 2X 10 in DMEM medium containing 10% FBS⁵cells/well density were plated into 12-well cell culture plates at 37 ℃ in 5% CO₂Culturing the culture box until the cell density reaches about 90%, discarding the culture medium, taking 500 mu L BHK-21 cell transfection supernatant, covering the supernatant on Marc-145 cells, incubating for 1.5h, discarding the supernatant, adding DMEM culture medium containing 2% FBS, and continuing culturing. Cell morphology was observed daily until the appearance of typical lesions (fig. 2). After 3-4 days of culture, cell supernatants were collected and subjected to IFA assay. As shown in figure 3, the PRRSV anti-N protein monoclonal antibody 15A1 is detected, the rescued virus (rHLJB1-M-Ame3) can see specific red fluorescence, the parental strain and a negative control group do not generate fluorescence, and the success of the rescue of the rHLJB1-M-Ame3 infectious clone virus on Marc-145 cells is verified. Meanwhile, rHLJB1-M-Ame3 infectious clone virus can also be successfully propagated on PAM cells (FIG. 4). rHLJB1-M rescued in the same way was still only able to infect primary PAM cells, but not Marc-145 cells (FIGS. 2-6).

1.5 determination of the proliferation activity of rHLJB1-M-Ame3 on Marc-145;

marc-145 cells were cultured at 2X 10 in DMEM medium containing 10% FBS⁵cells/well density were plated into 12-well cell culture plates at 37 ℃ in 5% CO₂The culture chamber of (2%) was cultured until the cell density reached about 90%, the medium was discarded, changed to DMEM maintenance medium (600 μ L) with 2% FBS, and the cell density was changed to 1: 20 (30. mu.L), the supernatants were discarded at 2hpi (hours post infection), 24hpi, 48hpi, 72hpi, 96hpi, 120hpi and 144hpi, respectively, and the cells were collected and total RNA was extracted using TRIpure Reagent. After the concentration of the total RNA is measured by the NanoDrop, 500ng of RNA is taken for reverse transcription. mu.L of the obtained cDNA was used for real-time PCR detection of PRRSV1 ORF7 gene. The specific reaction system and amplification procedure were as follows:

TABLE 4 detection of the reaction systems

cDNA	2×EX Taq	Primer (10. mu.M)	Probe (10 μ M)	H₂O
					1μL	20μL	0.5μL	0.4μL	Make up to 20 mu L

TABLE 5 detection reaction procedure

The dynamic proliferation of the rHLJB1-M-Ame3 engineered virus on Marc-145 is shown in FIG. 5. Only the rHLJB1-M-Ame3 engineered virus can replicate and propagate on Marc-145 cells, while neither the rHLJB1 nor the rHLJB1-M infectious cloned virus can propagate on Marc-145 cells.

1.6rHLJB1-M-Ame3 infects Marc-145 plaque assay;

marc-145 cells were cultured at 2X 10 in DMEM medium containing 10% FBS⁵cells/well density were plated into 12-well cell culture plates at 37 ℃ in 5% CO₂Culturing in the incubator until the cell confluence reaches 100%, discarding the culture medium, adding 600 μ L virus solution diluted in ten-fold gradient, incubating for 2h, and adding 2%FBS and 1 × DMEM low melting point agarose gel cover. Inverted at 37 deg.C and 5% CO₂The incubator of (1) was cultured for 7 days. The agar surface was then covered with 4% paraformaldehyde and fixed in a refrigerator at 4 ℃ overnight. Excess 4% paraformaldehyde was discarded, agar was carefully removed, crystal violet was added for staining for 1h, and plaque morphology was observed after tap water washing, as shown in fig. 6. Only the rHLJB1-M-Ame3 engineered virus could produce plaques after infecting Marc-145 cells, while neither the rHLJB1 nor the rHLJB1-M infectious cloned virus could infect Marc-145 cells, but did not produce plaques.

Example 2

A modified virus rHLJB1-M-Ame3 piglet inoculation test and safety and immune efficacy evaluation thereof;

1. a modified virus rHLJB1-M-Ame3 piglet inoculation test;

selecting 13 PRRSV, PRV (pseudorabies virus), PCV (circovirus), PEDV (porcine epidemic diarrhea virus) and other important pathogen negative pigs, and adding 10 in 2mL⁵TCID₅₀Inoculating 5-head 6-week-old piglets with 2mL of 10-containing rHLJB1-M-Ame3 virus solution⁵TCID₅₀The HLJB1 virus liquid is inoculated to 5 piglets. Another 3 piglets were inoculated with 2mL of DMEM medium. Sera were collected at 3, 7, 11, 14, 21dpi (days post infection) and assayed for viral content by qRT-PCR, as shown in figure 7. Piglet body temperature changes were monitored daily as shown in fig. 8. Piglets were monitored weekly for weight change as shown in fig. 9. The rHLJB1-M-Ame3 virus can proliferate in pig to produce viremia, but does not cause body temperature rise and influence weight gain. Furthermore, obvious clinical symptoms or death are not caused, and the modified virus is preliminarily shown to have better safety.

2. Detecting the content of the antibody in the serum of the inoculated pig by an in vitro neutralization test;

to determine the ability of the rHLJB1-M-Ame3 virus to induce a protective immune response in swine, a neutralizing antibody assay was performed on collected serum samples using a virus neutralization assay. Serum neutralization activity against rHLJB1-M-Ame3 virus was determined on Marc-145 cells. The result shows that the rHLJB1-M-Ame3 inoculated pig can induce the generation of neutralizing antibody, and the modified virus can induce pig body to generate protective immune response. Specific results are shown in the following table.

TABLE 6 serum antibody content of vaccinated pigs

It should be understood that the above examples are only for clearly illustrating the technical solutions and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Yangzhou university

Culture and application of <120> Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome virus

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20013

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 60

gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 120

agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 180

ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 240

cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 300

gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 360

cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 420

gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 480

ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 540

acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 600

ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 660

aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 720

aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccttaat aagatgatct 780

tcttgagatc gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg 840

cagggcggtt tttcgaaggt tctctgagct accaactctt tgaaccgagg taactggctt 900

ggaggagcgc agtcaccaaa acttgtcctt tcagtttagc cttaaccggc gcatgacttc 960

aagactaact cctctaaatc aattaccagt ggctgctgcc agtggtgctt ttgcatgtct 1020

ttccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg actgaacggg 1080

gggttcgtgc atacagtcca gcttggagcg aactgcctac ccggaactga gtgtcaggcg 1140

tggaatgaga caaacgcggc cataacagcg gaatgacacc ggtaaaccga aaggcaggaa 1200

caggagagcg cacgagggag ccgccagggg gaaacgcctg gtatctttat agtcctgtcg 1260

ggtttcgcca ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc 1320

tatggaaaaa cggctttgcc gcggccctct cacttccctg ttaagtatct tcctggcatc 1380

ttccaggaaa tctccgcccc gttcgtaagc catttccgct cgccgcagtc gaacgaccga 1440

gcgtagcgag tcagtgagcg aggaagcgga atatatcctg tatcacatat tctgctgacg 1500

caccggtgca gccttttttc tcctgccaca tgaagcactt cactgacacc ctcatcagtg 1560

ccaacatagt aagccagtat acactccgct agcgctgagg tctgcctcgt gaagaaggtg 1620

ttgctgactc ataccaggcc tgaatcgccc catcatccag ccagaaagtg agggagccac 1680

ggttgatgag agctttgttg taggtggacc agttggtgat tttgaacttt tgctttgcca 1740

cggaacggtc tgcgttgtcg ggaagatgcg tgatctgatc cttcaactca gcaaaagttc 1800

gatttattca acaaagccac gttgtgtctc aaaatctctg atgttacatt gcacaagata 1860

aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg 1920

ttatgagcca tattcaacgg gaaacgtctt gctcgaggcc gcgattaaat tccaacatgg 1980

atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa 2040

tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta 2100

gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc 2160

ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg 2220

cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata 2280

ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc 2340

cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt 2400

tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga 2460

aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct 2520

cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag 2580

tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt 2640

ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata 2700

aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt 2760

aacactggca gagcattacg ctgacttgac gggacggcgg ctttgttgaa taaatcgaac 2820

ttttgctgag ttgaaggatc agatcacgca tcttcccgac aacgcagacc gttccgtggc 2880

aaagcaaaag ttcaaaatca ccaactggtc cacctacaac aaagctctca tcaaccgtgg 2940

ctccctcact ttctggctgg atgatggggc gattcaggcc tggtatgagt cagcaacacc 3000

ttcttcacga ggcagacctc agcgctcaaa gatgcagggg taaaagctaa ccgcatcttt 3060

accgacaagg catccggcag ttcaacagat cgggaagggc tggatttgct gaggatgaag 3120

gtggaggaag gtgatgtcat tctggtgaag aagctcgacc gtcttggccg cgacaccgcc 3180

gacatgatcc aactgataaa agagtttgat gctcagggtg tagcggttcg gtttattgac 3240

gacgggatca gtaccgacgg tgatatgggg caaatggtgg tcaccatcct gtcggctgtg 3300

gcacaggctg aacgccggag gatcccgatg tacgggccag atatacgcgt tgacattgat 3360

tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 3420

agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 3480

gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 3540

gacgtcaatg ggtggactat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 3600

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 3660

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 3720

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 3780

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 3840

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 3900

ggcgtgtacg gtgggaggtc tatataagca gagctcgggc gcgcctacat gatgtgtagg 3960

gtattccccc tacatacacg acacttctag tgtttgtgtg ccttggaggc gtgggtatag 4020

ccccgcccca ctctttggcc cctgttctag cccaacaggt atccttctcc ctcggggcga 4080

gcgtgccgcc tgctgctctc ttgcagcggg aaggacctcc cgagtatttc cggagagcac 4140

ctgctttacg ggatctccac cctttaacca tgtctgggac gttctcccgg tgcatgtgca 4200

ccccggctgc tcgggtattt tggaacgccg gccaagtctt ttgcacacgg tgtctcagtt 4260

cacggtctct tctccctttg gaacttcagg acgctgacct tggtgtagtc ggcttgtttt 4320

acaagcccaa ggacaagctt cattggaaag tccctattgg tattcctcag atagaatgca 4380

ccccgtccgg gtgctgttgg ctctcggcca tttttcccat cgcgcgcatg acctccggca 4440

attacaactt cctccagcga ctcgtgaagg tggctgatgt tctgtaccga gatggttgct 4500

tggcaccacg acatcttcat gaactccaag tttatgagcg cggctgcagc tggtacccga 4560

tcacagggcc cgtgcccgga atgggtttgt ttgcgaactc catgcatgtg tctgaccagc 4620

cgttccctgg cgccacccat gtattgacca actcgccttt gcctcaacgg gcttgtcggc 4680

agccattttg tccatttgag gaggctcatt ccgacgtgta taggtgggag gggtttgtgg 4740

ttttcataga ctccaccccc aacggccggc ctcgtatgat gtggacaccg gggtctaatg 4800

attcagctgc cttggaagaa ctgccgtctg aactgaggtg tcaagccaag atccttattc 4860

gtagctttcc tgcccaccac cctgtcgacc tcgctgactg ggagctcact gagtcccccg 4920

agcatggttt ttctttcagc gtgtctcatc cctgtgggta catcgcccaa aaccccaaca 4980

cgtttgacgg caagtgctgg ctttcctgct ttttggacct gccggccgat gtatggcgtc 5040

acgaggagtt tctagccgac accttcggtt atcaaaccaa gtggggcgtg catggtaagt 5100

acctccagcg taggcttcag attcgtggca tgcgtgctgt agtcgatcct gatggtccta 5160

ttcacgtgga ggcactgtct tgcccccagt cttgggttag gcacctgact ttggacgacg 5220

aagttactcc aggattcgtt cgtctggcat ccctccgcat tgtgtcgaac acagagccca 5280

ccacttcccg gatctttcgt ttcggagcac ataaatggta tggtgctgcc ggcaagcggg 5340

ctcgcgccaa gcgtgccgct aggagtggga aggatccagc tcccaccccg gaggctgctt 5400

tgcctgcctc cgcttgcagc ggggaagacc gcggagtcat cacctactct ccacctgcag 5460

acggatcttg tggttggcat gtcctcggtg ccataatgaa ccggatgatg aatgatgact 5520

tcacgtcccc cctgactccg tacaacagac cagaggatga ttgggcttct gattatgatc 5580

ttgctcaggc gattcaatgc ctaaaactgc ctgctgccgt ggttcgaaat cgcccttgcc 5640

ccaacgccaa gtacctcata agactcaacg gggttcactg ggaggtagag gtgagagttg 5700

gaatggcccc tcgctccctt tctcgtgaat gcgtggttgg cgtctgctct gacggctgcg 5760

ccgcgctgcc ctatccagca gacgggctac ccaaacgtgc actcgaagcc ttggcgtctg 5820

cttacagact gccctctgat tgtgtctgct ctggcattgc cgattttctt gctgatccac 5880

ctcctcagga gttgtggacc cttgacaaaa tgctgacctc tccgtcacca gagcggaccg 5940

gtttctctag tttgtatagg ttactattgg aggttgttcc gcagaaatgc ggtaccacag 6000

agggggcttt cgtctatgct gttgagagga tgttaaagga ttgcccgagc tccaaacaag 6060

ccatggccct tctggcgaaa attaaagtcc catcctcaaa ggccccgtct atatctctgg 6120

acgagtgctt ccctacggat gttccagtgg actccgagtc agcgtttcag gaaaggcccc 6180

aaagttctgg tgctgctgtt gtcctgtgtt caccagacat gaaagtgttc agggaagcag 6240

ccccagaaga ggttcaagaa ggtggccaca tggccgtccg ctctacactc cttgccgatg 6300

atcttaacaa tgggcaggta cgggtggttg ctgacgcgca actaaagcta ggcagttatg 6360

acttggcggt cgggaatacc cacggaggtg taccgacttt agctagtcca accaacctag 6420

cagatgggaa tttgctcccc tcggactcca tgaaagggaa cgtgcccaat gattgggagg 6480

acgaaccact ggatttgtcc caaccagcac tagcgaccac aacgaccctt gtgagagagc 6540

aaacacccga caatccaagt cctggcgccg gtgccctccc tgtcaccatt cgaggatttg 6600

tcccggcaag gcctacaccc cgtcgtgttg agtactgcgg cacggggtcg ggcgacagca 6660

gttcgcctct ggatcagtcc gatgcgcaaa ccacggacca gcctttaaat ttatccctgg 6720

ccgcttggcc agtgaaggcc accgcgtctg accccggctg ggtctacggt aggcgtgagc 6780

ctgtttttgt aaagcctcgg ggtgctctct ctgatggcga ctcagtcctt cagtttggag 6840

agttttccga aaccggctct atcattgaga ttgaccggac aaaagacatt ccagtggttg 6900

atgcccccaa cgacttgacg gtttcgaacg aaactctctc tgggagcgat cctttcgaat 6960

tcgccgaact caaacgcccg cgtttctccg ctcaagccct aattgaccga ggcggcccac 7020

tagccgatgt ccatgcaaag ataaagaacc gggtgtatga acagtgcctc caagcatgtg 7080

agcccggtag tcgtgcaact ccggccacca aggagtggct cgacaaaatg tgggacaggg 7140

tggacatgaa aacttggcgt tgcacctcgc agttccaagc tggtcgcatc cttgcgtccc 7200

tcaacttcct ccctagcatg attcaagaaa caccacctcc tgttcccagg aagggccgga 7260

ccagtgacaa cactgacttg aagcggttga tggcacagtg ggatagaaaa ttgagtgttg 7320

cccctcccca gaaattggct gggccagtgc ttggccaagc cgtccattcg cctgcgaaca 7380

cccaacaaga agaggtcacc ccctctgacg gaccacccta tgcaccgggt tttctgaatc 7440

gggtgagcgc gggcgggagt tggaaagacc ttatgtcttc cagtgcccgt cttgcggggt 7500

ctatcagtca gcgcattatg acatgggttt ttgaaactta ctcccatctc ccagcctttg 7560

cactcgcact attctcgcca cggggcccta tggctccagg tgattggttg tttgcaggtg 7620

ttgttttact tgctctcctg ttatgtcgcc tttacccgat actcgggtgc cttcccttat 7680

tgggtgtctt ttctggttct ttgcggcgtg ttcgtctggg tgtttttggt tcttggatgg 7740

cttttgctgt atttttattc tcgactccat ccgatccagt cggttcttct tgtgaccacg 7800

attcgccgga gtgtcatgct gagcttttgg ctcttgagca gcgccaactt tgggaacctg 7860

tgcgtggcct tgtggttggt ccctcaggtc tcttatgtgt cattcttggt aagttactcg 7920

gtgggtcacg ttatctctgg catattttct tacgtttatg catgcttgcg gatttggccc 7980

tttctcttgt ttatgtggtg tcccaggggc gttgtcacaa gtgttgggga aagtgtataa 8040

ggacagctcc tgcggaggtg gctctcaatg tgttcccttt cttgcgtgct acccgtgcct 8100

ccctcgtgtc cttgtgcgat cgattccaag cgccaaaagg ggttgatcct gtgcacttgg 8160

caacaggttg gcgcgggtgc tggcgcggtg agagccccat tcatcaaccg caccaaaagc 8220

ccatagctta tgccaatttg gatgaaaaga agatatctgc ccaaacggtg gttgccgttc 8280

cgtacgatcc tagtcaggcc atcaaatgcc taaaagttct gcaagcggga ggggctatcg 8340

tggaccaacc cacacctgag gtcgtccgtg tgtccgagat ccctttctca gccccatttt 8400

ttccaaaggt tccagtcaac ccagattgta gggtcgtggt agattcggac acttttgtgg 8460

ctgcagttcg ctgcggttac tcgacggcac aactggtctt aggccagggc aactttgcca 8520

agttaaatca gatcccctcc ggaagccccg tctctaccaa aactactggt ggggcctctt 8580

acacccttgc tgtggctcag gtgtctgtgt ggactctcgt ccatttcacc ctcggtcttt 8640

ggttcacatc gcctcaagtg tgtggccgag gaacttctga tccatggtgt tcaaatcctt 8700

tttcatatcc tgcctatggc cctggaacag tgtgctcctc tcgcctttgt gtgtccgccg 8760

acggggtcac tctgccactg ttctcagcag tggcacaact ctccggtaga gaggtgggga 8820

tttttatcct ggtgctcgtt tccctgattg ccctggccca ccgtttggct cttaaggcag 8880

acatgttagt ggtcttctta gccttttgtg cttacgcctg gcccatgagt tcctggctaa 8940

tctgcttctt tcctatgttc ttgaagtggg tcacccttca ccctctcact atgctttggg 9000

tgcactcatt cttggtgttt tgtctgccag cagccggcgt cctctcatta gggataaccg 9060

gccttctctg ggcagttggc cgcttcaccc aggtcgccgg aattatcaca ccttatgaca 9120

tccaccagta cacctctgga ccacgtggtg cagccgccgt ggccacggcc ccagaaggca 9180

cttacatggc cgccgtccgg agagctgccc taactggacg aaccctgatc ttcacaccat 9240

ctgcggttgg atcccttctt gaaggtgctt tcaggactca caaaccctgc ctcaacaccg 9300

tgaatgttgt aggctcttcc ctaggttccg gggggatttt cactattgat ggcagaagaa 9360

ctgttgtcac tgccgcccac gtgttgaacg gcaacacagc tagggtcacc ggcgattcct 9420

acaaccgcat gcacactttc aaaactaatg gtgattatgc ctggtcccat gctgacgact 9480

ggcggggcgt tgcccctgtc gttaatgtcg cgaaggggta ccgcggtcgt gcctactggc 9540

aaacgtcaac tggtgtcgaa cccggcattg ttggggaagg gtttgccttc tgcttcacca 9600

attgtggcga ttcggggtca cctgtcattt cagaatctgg tgatcttgtt ggaatccaca 9660

ccggttcaaa caaactcggc tctggccttg tgacaacccc tgaaggggag acctgctcca 9720

tcaaagaaac caagctctct gatctctcca ggtattttgc aggcccaagc gtccctcttg 9780

gggatataaa attgagtcca gccatcatcc ctgacgtaac atccattccg agcgacttgg 9840

catcgctcct agcctccgtc cctgtgatgg aaggcggcct ctcgactgtt caacttttgt 9900

gtgtcttttt cctcctctgg cgtatgatgg gccatgcctg gacacccatt gttgccgtgg 9960

gctttttttt gctgaatgaa attcttccag cagttttggt ccgagccgtg ttttcttttg 10020

cgctctttgt gcttgcatgg gtcaccccct ggtctgcaca ggtgttgatg atcagactcc 10080

tcacggcatc tcttaatcgc aacaagcttt ctctggtgtt ttacgcactc gggggtgtcg 10140

tcggtttggc tgctgaaatt gggaccttcg ctggtagatt gtctgaattg tctcaagctc 10200

tttcaacata ctgcttcttg cctagggttc ttgctgtgac cagttatgtt cctatcatca 10260

tcatcggtgg actccatgcc cttggcgtaa ttttgtggct attcaaatac cggtgcctcc 10320

acaacacgtt agttggtgat gggagtttct caagcgcctt tttcctacgg tattttgcag 10380

agggtaacct cagaaagggt gtttcacagt cctgcggcat gaataacgag tccctgacag 10440

ctgctttagc ttgcaagttg tcgcaggctg acctcgaatt tttgtccaac ttgacgaact 10500

ttaagtgctt cgtgtctgct tcaaacatga aaaatgctgc cggtcagtat gttgaagcgg 10560

cgtatgccaa ggcccttcgc caagagctgg cttccctggt tcaggttgat aagatgaaag 10620

gggtcttgtc caaacttgag gcctttgctg aaacagccac cccgtccctc gacacaggtg 10680

acgtgatcgt tctgcttggg caacaccctc atggatcaat ccttgacatt aatgttggga 10740

ctgaaaggaa aactgtgtct gtgcaagaga cccggagcct aggcggttcc aaattcagcg 10800

tctgcactgt cgtgtctaac acacccgtgg acgccttaat tgatatccca ctccaaacac 10860

caacccctct ttttgaaaac ggtccgcgtc atcgtggcga ggaggatgat cttagagtag 10920

agagggcgaa gaagcactgt gtgtccctcg gcttccacaa catcaatggt aaagtctact 10980

gcaagatttg ggacaagtct accggtgaca ccttctacac ggacgattcc cggtatactc 11040

aggactatgc ttttcaggac aggtcagccg actacagaga cagggactat gaaggtgtgc 11100

aaaccgcccc tcaacaggga tttgatccga agtctgaaac ccctgttggc actattgtga 11160

tcggcggaat tacgtataac aggtatctga tcaaaggtaa ggagattctg gtccccaggc 11220

ctgacaactg tctcgaagct gccaggttgt cccttgagca agctctcgct ggaatgggcc 11280

aaacttgcaa ccttacagct gccgaattgg aaaagctgaa acgcatcatc agtcaactcc 11340

aaggtttgac caccgagcag gctttaaact gttagccgcc agcggcttga cccgctgtgg 11400

ccgcggcggc ctagttgtga ctgaaacggc ggtgaaaatt gtaaagtacc acagcagaac 11460

cttcacctta ggctctttag atttaaaagt tacttccgaa gtggaggtga agaaatcaac 11520

tgagcagggt cacgctgttg ttgcaaactt gtgttccggc gtcatcttga tgagacctca 11580

cccaccgtct ctcgttgacg ttctcttgaa acccggactc gacacaacgc ctggcattca 11640

accgggacat ggggccggga acatgggcgt ggacggctct atttgggact ttgaaactac 11700

acccacaaaa gcagaactcg agttgtccaa gcagataatt caagcctgtg aagttaggcg 11760

cggagacgcc ccgaatctcc aactccccta caagctctat cctgtcagag gggatcctga 11820

gcggcataag ggccgcctta tcaataccag gtttggagac ctgccttaca aaactcctca 11880

agacactaag tccgccatcc atgcggcttg ttgcctgcac cccaacgggg cccctgtgtc 11940

tgatggcaaa tccacattag gcaccactct gcaacatggt ttcgagcttt atgttcccac 12000

agtgccctat agtgtcatgg agtaccttga ttcacgccct gacacccctc ccatgttcac 12060

taaacatggc acttccaggg ccgctgcaga agacctccaa aaatatgacc tatccaccca 12120

aggatttgtc ctgcctgggg tcctacgctt ggtgcgcaaa ttcatctttg gccatgttgg 12180

taaggcaccg ccattgttcc tcccatcaac ctatcccgct aagaactcta tggcagggat 12240

taacggccaa agattcccaa caaaggacgt ccagagcata cctgaaattg atgaaatgtg 12300

tgcccgcgcc gtcaaggaga attggcaaac cgtgacacct tgcactctca agaaacagta 12360

ctgctccaag cccaaaacca ggaccatcct gggcaccaat aacttcattg ccttggctca 12420

cagatcggca cttagtggcg tcaccagtgc attcatgaag aaggcttgga agtccccaat 12480

tgccttgggg aaaaacaagt tcaaggagct gcattgtact gtcgccggca ggtgtcttga 12540

ggctgacttg gcctcctgtg atcgtagcac cccagccatt gtaagatggt ttgtcgccaa 12600

cctcctgtat gaacttgcag gatgtgagga gtacttgcct agctatgtac tcaactgctg 12660

tcatgacctt gtggcaacac aggatggtgc cttcacaaaa cgcggtggcc tgtcgtccgg 12720

ggaccctgtc accagtgtat ccaataccgt atattcactg gtaatctatg cccagcacat 12780

ggtattgtca gccttgaaaa tgggtcatga aattggtctt aagttcctcg aggagcagct 12840

caaattcgag gacctccttg aaattcagcc catgttagta tactctgacg atcttgtctt 12900

gtatgctgaa aggcccactt ttcccaatta ccattggtgg gtcgagcacc ttgacttgat 12960

gctgggcttc aaaacggacc caaagaaaac tgttataact gacaaaccca gcttcctcgg 13020

ctgcaggatt gaggcagggc gacagttggt ccccaaccgc gaccgcatcc ttgctgccct 13080

tgcctatcac atgaaggcgc agaacgcctc agaatattat gcgtccgctg ccgcaatcct 13140

gatggattcg tgcgcttgca ttgaccatga ccctgagtgg tatgaggacc tcatctgtgg 13200

tattgcccgg tgcgctcgcc aagatggcta tagtttcccg ggcccggcat ttttcatgtc 13260

catgtgggag agactgaaaa gtcataatga agggaaaaaa ttccgtcact gtggcatctg 13320

cgacgccaag gccgaccatg cgtccgcctg tggacttgat ttgtgcttgt tccactcgca 13380

ttttcatcag cactgccctg tcactctgag ctgcggccat catgccggtt ctgaggagtg 13440

tccgcagtgt cagtcaccgg ttggggctgg tagatctcct ctcgatgctg tgctgaaaca 13500

aatcccgtac aagcctcctc gtactgtcat catgaaggtg gacaataaaa caacggccct 13560

tgatccgggg agatatcaat cccgtcgagg ccttgtggca gtcaagaggg gtgttgcagg 13620

caatgaagtt gaccttgctg atggagacta ccaggtggtg cctcttttgc cgacttgcaa 13680

agatataaac atggtgaagg tggcttctaa tgtgctactc agcaagttca tagtagggcc 13740

accaggctcc ggaaagacca cctggttgct aagtcaagtc caggacgatg atgtcattta 13800

tacacccacc catcagacta tgtttgatat agtcagtgct ctcaaagttt gcaggtattc 13860

cattccaggg gcttcagggc tccctttccc gccacctgcc aggtccgggc cgtgggtcag 13920

gctcgtcgcc agtgggcacg tccctggccg agtatcatac ctcgatgagg ctgggtattg 13980

taatcatctg gacattctca gactgctttc taaaacaccc cttgtgtgtt taggtgacct 14040

tcagcaactc caccctgtcg gctttgattc ctactgttat gtgtttgatc aaatgcctca 14100

gaagcaattg actactattt acagatttgg tcccaacatc tgtgcggcca tccagccttg 14160

ttacagagaa aagcttgaat ctaaggctag aaacaccaga gtggtcttca ccacacggcc 14220

tgtggccttc ggtcaggtgc tgacaccgta tcacaaagat cgcatcggtt ctgcgataac 14280

catagattca tcccaggggg ccacttttga cattgtgaca ctgcatttgc catcgccaaa 14340

gtcattaaat aaatcccgag cacttgtggc tatcactcgg gcaagacacg ggttgtttat 14400

ttatgacccc cataaccagc ttcaggagtt tttcaactta acccccgagc gtactgattg 14460

caaccttgtg ttcagctgtg gggatgagct ggtagttctg gacgcagaca atgcagtcac 14520

aactgtagcg agggccctag aggcaggccc gtcccgattt cgagtatcag acccgaggtg 14580

taagtccctc ttagccgctt gttcggccag tctggaaggg agttgcatgc cgctaccaca 14640

ggtggcacat aacctagggt tttacttttc cccagacagt ccggcatttg cacctctgcc 14700

aaaagagctg gcgccacatt ggccagtggt tacccaccaa aataatcagg cgtggcctga 14760

tcgactcgtc gccagcatgc gtccaattga tgcccgctac agtaagccaa tggtcggtgc 14820

agggtatgtg gttggaccgt ccacctttct tggcactcct ggtgtagtgt cgtattatct 14880

cacactatac atcaagggtg agccccaggc cttgccagaa acactcgttt caacaggacg 14940

tatagccaca gattgtcggg aatatcccga cgcggctgag gaggaggcag caaaagaact 15000

cccccacgcg tttatcggtg atgtcaaagg caccacggtt ggggggtgtc atcacattac 15060

atcaaaatat ctacccaggt ccctgcctaa agactctgtt gccgtggttg gggtaagttc 15120

gcccggcagg gctgccaaag ccgtgtgcac tctcactgac gtgtacctcc ccgaacttcg 15180

accatatttg caacctgaga cggcgtcaaa atgctggaaa ctcaaattag acttcaggga 15240

tgtccggctg atggtttgga aaggagccac cgcctacttc caattggaag ggctcacgtg 15300

gtcggcgttg cccgactatg ctaggtttat tcaactcccc aaggatgctg ttgtatacat 15360

cgatccgtgt ataggaccgg caacggccaa ccgtaaggtt gtgcgaacca cggactggcg 15420

ggccgaccta gcagtgacac cgtatgatta cggcgcccag aacatcttga caacagcctg 15480

gttcgaggac ctcggaccgc agtggaagat attggggttg cagcccttca ggcgagcact 15540

tggtcttgag aacaccgagg actgggcaat cctagcacgc cgcatgaatg acggcaagga 15600

ctacactgat tataactgga gctgtgttcg tgagcgccca cacgccgttt acgggcgtgc 15660

tcgtgaccac acgtatcatt tcgctcctgg tacagaacta caggttgagc taggtaaacc 15720

tcggctgcta cctgagcaag tgccgtgaac tcggagcgat gcgatggggt cgctgtggag 15780

taaaatcagc cagctgtttg tggacgcttt caccgagttc cttgttagtg tggtcgatat 15840

tgtcattttc cttgccatac tgttcgggtt cacagtcgca gggtggctac tggtctttct 15900

ttttagagtg gtttgctccg cgtttctccg ttcgcgctct gccgttcact ctcccgaact 15960

atcgaagatc ctatgaaagc ttgttgcccc attgcagacc ggatgtccca caatttgcat 16020

tcaagcatcc attaggtata ctttggcaca tgcgagtttc ccacctgatt gatgaaatgg 16080

tctcccgtcg tatttaccag accatggaac attcaggcca agcagcctgg aaacaggtgg 16140

tcagtgaagc caccctcacg aagttgtcaa ggctcgacgt agtcactcac tttcaacacc 16200

tggccgcagt ggaggcagat tcttgccgtt ttctcagctc acgactcgtg atgctaaaaa 16260

accttgccgt tggcaatgtg agcttacagt acaacaccac gttggaccgc gttgagctca 16320

ttttccccac gccaggtacg aggcccaagt tgaccgactt cagacaatgg ctcatcagtg 16380

tgcacgcttc cattttttcc tctgtggctt catctgttac cttgttcata gtgttttggc 16440

ttcgaattcc agccgtacgc tatgtttttg gtttccattg gcccacggca acacatcatt 16500

cgagctaacc atcaactaca ccatatgtat gccctgctct accagccaag cggctagcca 16560

aagactcgag cccggtcgta acatgtggtg cagaataggg cacgacaggt gtgaggaacg 16620

tgaccatgat gagttgtcaa tgtccattcc gtcagggtac gagaacctca aacttgaggg 16680

ttattatgct tggctggcct ttttgtcctt ttcctacgcg gcccaatttc atccggagtt 16740

gttcggaata ggaaacgtgt cgcgcgtctt tgtggacaag cgacaccagt tcatttgcgc 16800

cgagcatgat ggacaaaatt caaccatatc taccggacac aacatctccg cattatatgc 16860

ggtgtattac catcaccaaa tagacggggg caattggttc catttggaat ggctgcggcc 16920

attcttttcc tcctggctgg tgctcaatat ctcatggttt ctgaggcgtt cgcctgtaag 16980

ccctgtttct cgacgcatct atcagatatt aagaccaaca cgaccgcggc tgccggtttc 17040

atggtccttc aggacatcaa ttgtctccga cctcacgggg tctcaacagc gcaagagaac 17100

attcccttcg gaaagccgtc ccaatgtcgc gaggccgtcg gtattcccca gtacattacg 17160

ataacggcta atgtgaccga tgaatcgtat ttgtacaacg cggacttgct tatgctttct 17220

gcgtgccttt tctacgcttc agaaatgagc gagaagggct tcaaggtcat cttcgggaac 17280

gtttccggcg ttgtttctgc ttgtgtcaat tttacagatt atgtggctca tgtaacccaa 17340

catacccagc agcatcatct ggtgattgat cacattcggt tgctgcattt cctgacacca 17400

tcaacaatga ggtgggctac aaccattgcc tgtttgttcg ccattctctt ggcgatatga 17460

gatgttctca caaattgggg cgtttcttga ttccgcactc ttgctcttgg tggctttttt 17520

tgttgtgtac cggcttgtct tggtcctttg ccgatggcaa cggcaacagc tcgacatacc 17580

aatacatata taatttgacg atatgcgagc tgaatgggac caattggttg tccagccatt 17640

ttgactgggc agtcgagacc tttgtgtttt acccggttgc cacccacatt ctttcgttgg 17700

gtttcctcac aacaagccat ttttttgatg cgctcggtct cggcgctgtt tccgctacag 17760

gatttgttgg caggcggtat gtacttagca gtgtgtacgg tgcttgtgct ctcgcagcgt 17820

tcgtatgttt tgtcatccgt gctgcaaaaa attgcatggc ttgccgttat gcccgcaccc 17880

ggtttaccaa ctttattgtg gacgaccggg ggaggatcca tcgatggaag tctccaatag 17940

tggtggagag attgggcaaa gctgaagtcg gtggcgacct cgtcaccatc aaacatgtcg 18000

ttctcgaagg ggttaaagct caacccttga cgagaacttc ggctgagcaa tgggaagcct 18060

agacgatttt tgcaacgatt ctaccgccgc acaaaagctt gtactggcct tcagcattac 18120

atatacaccc ataatgatat acgcccttaa ggtgtcacgc ggccgcctcc tggggttatt 18180

gcacatcttg atattcctga actgttcctt tacattcgga tatatgacgt atgtgcattt 18240

tcaatccacc aaccgcgtcg catttactct gggggccgtt gttgcccttt tatggggtgt 18300

ttacagcttc acagagtcat ggaagttcat tacttccaga tgtagattgt gttgcctagg 18360

ccggcgatac attctggccc ctgcccatca cgtagaaagt gctgcaggtc tccatccaat 18420

ctcagcgtct ggtaaccgag catacgctgt gagaaagccc ggactaacat cagtgaacgg 18480

cactctagta ccaggacttc ggagcctcgt gctgggcggc aaacgagctg ttaaacgagg 18540

agtggttaac ctcgtcaagt atggccggta aaaatcagag ccagaagaaa aagaagaata 18600

cagctctaat ggggaatggc cagccagtca atcaactgtg ccagttgctg ggtgcaatga 18660

tgaagtccca gcgccagcaa cctaggaaag gacaggcaaa aaagaaaaag tttgagaagc 18720

cacattttcc cctagctgct gaagatgaca ttcggcacca tctcacccag accgaacgtt 18780

ccctctgctt gcaatcgatc cagacggcct ttaatcaagg cgcaggaact gcgtcgcttt 18840

catccagtgg gaaggtcggt tttcaggttg agttcatgct gccggttgct catacagtgc 18900

gcttgattcg cgtgacttct acacccgcca gtcagggtgc agattaattt ggtagtcagg 18960

tgaatggccg cgattgacgt gtggcctcta agtcacctat tcaattaggg cgatcacatg 19020

ggggtcaaac ttaattaggc aggaaccatg tgaccgaaat taaaaaaaaa aaaaaaaaaa 19080

aaggccggca tggtcccagc ctcctcgctg gcgccggctg ggcaacattc cgaggggacc 19140

gtcccctcgg taatggcgaa tgggactcta gactgtgcct tctagttgcc agccatctgt 19200

tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 19260

ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 19320

tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 19380

tgcggtgggc tctatggtag agcgcacgaa tgagggccga caggaagcaa agctgaaagg 19440

aatcaaattt ggccgcaggc gtaccgtgga caggaacgtc gtgctgacgc ttcatcagaa 19500

gggcactggt gcaacggaaa ttgctcatca gctcagtatt gcccgctcca cggtttataa 19560

aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 19620

ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 19680

tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 19740

atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 19800

attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 19860

gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 19920

agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 19980

aaagttctgc tatgtggcgc ggtattatcc cgt 20013

<210> 2

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agctcgggcg cgcctacatg atgtgtaggg tatt 34

<210> 3

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcctgac taggatcgta cggaacggca accaccgt 38

<210> 4

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acggtggttg ccgttccgta cgatcctagt caggccat 38

<210> 5

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agcacagcat cgagaggaga tctaccagcc ccaaccggt 39

<210> 6

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

accggttggg gctggtagat ctcctctcga tgctgtgct 39

<210> 7

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ataagcaagt ccgcgttgta caaatacgat tcatcggt 38

<210> 8

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

accgatgaat cgtatttgta caacgcggac ttgcttat 38

<210> 9

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agcgaggagg ctgggaccat gccggccttt tttttttttt ttttttttaa tttcggtcac 60

atggttcct 69

<210> 10

<211> 87

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acagtctaga gtcccattcg ccattaccga ggggacggtc ccctcggaat gttgcccagc 60

cggcgccagc gaggaggctg ggaccat 87

<210> 11

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acaatttgca ttcaagcatc cattaggtat actttggcac at 42

<210> 12

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgtgccaaa gtatacctaa tggatgcttg aatgcaaatt gt 42

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

agttgatggt tagctcgaat gatgtgttgc 30

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gcaacacatc attcgagcta accatcaact 30

Claims

1. A recombinant plasmid is used for preparing Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome modified virus, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 1.

The Marc-145 cell adapted porcine reproductive and respiratory syndrome 1 modified virus rHLJB1-M-Ame3, wherein the rHLJB1-M-Ame3 virus is rescued and prepared from the recombinant plasmid of claim 1.

3. The method for constructing the Marc-145 cell-adapted type 1 porcine reproductive and respiratory syndrome-modified virus rHLJB1-M-Ame3, which is characterized by comprising the following steps of:

4. The method for constructing the Marc-145 cell-adapted type 1 porcine reproductive and respiratory syndrome modified virus rHLJB1-M-Ame3, which is characterized in that the infectious clone virus rHLJB1 of the PRRSV1 wild strain is as follows:

inserting five single restriction enzyme sites SgsI, Pfl23II, BglII, Bsp1407I and XbaI into a pACYC177 vector through gene synthesis and homologous recombination by using a pACYC177 low-copy vector, and then constructing the infectious clone virus rHLJB1 by using the recombinant vector; the rHLJB1 comprises a genome full-length cDNA sequence of a PRRSV1 wild strain HLJB1, wherein a cytomegalovirus eukaryotic promoter sequence is added to the 5 'end of the full-length cDNA sequence, and a hepatitis D virus ribozyme sequence and a bovine growth hormone polyadenylation signal transcription termination sequence are added to the downstream of the tail of the 3' end Poly (A).

5. The construction method according to claim 3 or 4, characterized in that the method comprises the following steps:

1) synthesizing pUC57-Synthesis-Amervac-ORF2-4 plasmid;

2) designing a primer;

3) construction of an infectious clone of HLJB1-M-Ame 3;

4) rescue of infectious clonal virus of rHLJB1-M-Ame 3.

6. The method for constructing a recombinant vector according to claim 5, wherein the primers designed in step 2) are as follows:

application of Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome modified virus rHLJB1-M-Ame3 in preparation of novel genetic engineering vaccines.