CN102321661A

CN102321661A - Eukaryotic expression recombinant plasmid and construction method thereof and Vero cell line stably expressing the plasmid

Info

Publication number: CN102321661A
Application number: CN 201110257669
Authority: CN
Inventors: 赵建军; 闫如勋; 闫喜军
Original assignee: Institute Special Animal and Plant Sciences CAAS
Current assignee: Institute Special Animal and Plant Sciences CAAS
Priority date: 2011-09-01
Filing date: 2011-09-01
Publication date: 2012-01-18

Abstract

The invention relates to the technical field of bioengineering, and particularly relates to an eukaryotic expression recombinant plasmid and a construction method thereof and a Vero cell line stably expressing the plasmid. The eukaryotic expression recombinant plasmid has the nucleotide sequence shown in SEQ ID NO:1. The Vero cell line stably expressing the plasmid can be used for high-efficiency separation of canine distemper virus virulent strains and the research of the pathogenic mechanism of canine distemper virus.

Description

Eukaryotic expression recombinant plasmid, construction method thereof and Vero cell line for stably expressing plasmid

Technical Field

The invention relates to the technical field of bioengineering, in particular to a eukaryotic expression recombinant plasmid, a construction method thereof and a Vero cell line for stably expressing the plasmid.

Background

Canine Distemper (CD) is a highly contagious disease of animals in canines and ferrets caused by Canine Distemper Virus (CDV). The disease is one of the most harmful infectious diseases in the dog industry and fur-bearing animal breeding industry, is typically characterized by anorexia, diphasic heat, conjunctivitis, gastroenteritis and nervous symptoms, can cause diseases of a large number of dogs, foxes, raccoon dogs and other animals, and has the morbidity and mortality rate of 30-80%. In recent years, the range of canine distemper virus infection hosts is increasingly expanded, and infections of canine distemper virus are reported in macaques of the order primates, seals of the family seal of the order finpodales, pandas, lions of large animals of the family felidae, wild dogs of africa, and the like. In China, with the great increase of the feeding amount of military dogs, police dogs, experimental dogs, fur-bearing animals such as foxes and raccoon dogs and the like and the increase of allopatric communication in recent years, the incidence rate and the fatality rate of canine distemper in dogs, foxes and raccoon dogs tend to increase, so that the canine distemper prevention and control are particularly important.

The virus separation technology is an important means for researching the pathogenic mechanism of the canine distemper virus, but the canine distemper virus with the envelope is not easy to survive in the environment, so that the canine distemper virus is difficult to be successfully separated by a common method. At present, the separation of canine distemper virus wild strains is generally carried out by a method of coculture of susceptible animal lung macrophages and Vero or MCDK and other continuous cell lines, but the method has the defects of complex operation, long time consumption, unstable virus titer obtained by separation and the like, and the separated canine distemper virus is likely to have virulence gene mutation in the process of adapting to cells due to the fact that the above continuous cells are canine distemper virus non-sensitive cells, so that the virus strains are weakened. Because canine distemper virus (especially virulent strain) lacks sensitive and applicable cells or cell lines during in vitro culture, the separation success rate of the canine distemper virus is low (generally lower than 20 percent), and the research on the aspects of the pathogenic biology and pathogenic mechanism of the canine distemper virus is seriously hindered.

In 2001, Tatsuo et al infected wild strain of CDV on CHO cells artificially expressing canine signaling lymphocyte activating molecule (SLAM is also called CD150), and experiments confirmed that SLAM of dogs is a receptor for CDV infection, and adsorption of CDV H protein to cell surface by recognition of SLAM initiated the infection process of virus. In 2003, the Vero cell line (Vero. DogSLAmTag) which is established by Seki et al and stably expresses canine SLAM is used for separating canine distemper virus from suspected canine distemper dogs more quickly and sensitively than Vero and B95a cells. Because the tag expressed in Vero.DogSLAmTag is the antigen epitope of the HA protein of the avian influenza virus, the positive Vero cell line identification needs to be carried out by means of an immunoblotting or indirect immunofluorescence method by means of a monoclonal antibody aiming at the epitope. Therefore, the methods used for the establishment and identification of such cell lines are cumbersome. In addition, because the expression level of canine SLAM in the positive Vero cell line is low, the virus separation sensitivity is not high, and the separation efficiency of canine distemper virus is directly influenced. In 2010, SLAM genes of foxes, raccoon dogs and minks were successfully cloned by Zhaojian army and the like, and all genes were confirmed to be receptors for canine distemper virus infection.

Therefore, a recombinant plasmid of the SLAM gene which is efficiently expressed and sensitive to virus tropism is constructed and is introduced into Vero cells to obtain a Vero cell line which stably expresses the SLAM gene, and the method has practical significance for quickly and sensitively separating the canine distemper virus and researching the pathogenic mechanism of the canine distemper virus.

Disclosure of Invention

In view of the above, the present invention provides a eukaryotic expression recombinant plasmid, a construction method thereof, and a Vero cell line stably expressing the plasmid. The eukaryotic expression recombinant plasmid has the nucleotide sequence shown in SEQ ID NO: 1. The Vero cell line can be used for efficiently separating canine distemper virus virulent strains and can be applied to the research of canine distemper virus pathogenic mechanisms.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a eukaryotic expression recombinant plasmid with the nucleotide sequence shown in SEQ ID NO: 1.

The eukaryotic expression recombinant plasmid consists of a raccoon SLAM gene (rSLAM) with the GeneBank accession number of EU678639, a fusion gene of a nucleotide sequence for coding 6 histidines (6 x his) and a Kozak sequence, Pcvv, IRES, EGFP and SV40pA, and is shown in figure 15. Wherein, the 5 'end to the 3' end of the plasmid are Pmvs, the fusion gene, IRES, EGFP and SV40pA in sequence, as shown in figure 13. Kozak sequence, a sequence present in eukaryotic mRNA, has the effect of enhancing the amount of protein expressed during initiation of translation. The optimal sequence of the upper and lower reaches of the initiation codon in the eukaryotic gene is purine base at the-3 position; the +4 position is G, and the A in the initiation codon AUG is at the +1 position.

Preferably, the Kozak sequence has a nucleotide sequence shown as SEQ No. 4.

The invention adopts a eukaryotic expression vector pIRES2-EGFP which is purchased from Clontech company in America, the eukaryotic expression vector pIRES2-EGFP is a binary expression vector, a target gene inserted in a Multiple Cloning Site (MCS) and the EGFP are co-expressed under a CMV promoter, the expression can be monitored (the expression condition of the target protein is reflected by observing the expression of green fluorescent protein under a fluorescence microscope), and the biological characteristics of the target protein can be well maintained.

The expression vector contains:

(1) a multi-cloning site MCS for cloning exogenous genes;

(2) allowing two encoding genes on one mRNA molecule to obtain proper internal entry sites IRES of the ribosome of the human encephalomyocarditis virus;

(3) using an enhanced green fluorescent protein gene EGFP;

(4) adding the SV40polyA signal region of the polyA tail to the mRNA molecule;

(5) pcvv models expression of later foreign genes and EGFP genomes. After Xho I and EcoR I double enzyme digestion, Pcvv, IRES, EGFP and SV40pA are obtained.

The sequence of the eukaryotic expression vector pIRES2-EGFP has a nucleotide sequence shown in SEQ No.5, and the circular map is shown in figure 14.

Wherein Pcvm has the amino acid sequence of SEQ ID NO: 6, IRES has the nucleotide sequence shown in SEQ ID NO: 7, EGFP has the nucleotide sequence shown in SEQ ID NO: 8, SV40pA has the nucleotide sequence shown in SEQ ID NO: 9.

The invention also provides a preparation method of the eukaryotic expression recombinant plasmid, which comprises the following steps:

step 1: the plasmid pMD18-T-rSLAM cloned with rSLAM is taken as a template, the fusion gene is obtained by amplification in the presence of an upstream primer and a downstream primer, the sequence of the upstream primer is shown as SEQ No.2, and the sequence of the downstream primer is shown as SEQ No. 3;

step 2: pIRES2-EGFP vector was digested to obtain Pcvm, IRES, EGFP and SV40 pA.

And step 3: taking the fusion gene: after enzyme digestion, the plasmid is connected with Pmv, IRES, EGFP and SV40pA in the step 2 to transform a recipient bacterium, and positive clones are identified to obtain the plasmid.

Taking the plasmid pMD18-T-rSLAM as a template and P with the sequence shown as SEQ No.2₁：5’-CCG

ATGGATTCCAGGGGCTTCCTC-3' as upstream primer, and P with sequence shown in SEQ No.3₂：5’-CCG

TCAGTGATGGTGATGGTGATGGCTCTCTGGGAACGTCAC-3' is a downstream primer (box is restriction enzyme cutting site, bold is Kozak sequence, underline is histidine label gene); PCR amplification is performed to obtain the target gene.

The reaction system can be: 1. mu.L (0.2 ng/. mu.L) of DNA template, 1. mu.L (20pmoL/L) of each of the upstream and downstream primers, 0.3. mu.L (5U/. mu.L) of Ex taq enzyme, 2. mu.L of dNTPs (2.5 mmol/L each), 2. mu.L of 10 XPCR buffer, 2.5. mu.L of ddH2O to 25. mu.L. The amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61.8 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; total extension at 72 ℃ for 10 min; storing at 4 deg.C for 10 min. The PCR product was recovered by 1% agarose gel, ligated into pMD18-T-simple vector, and subjected to restriction enzyme identification.

Preferably, the enzyme is Xho I and EcoR I double enzyme.

The raccoon SLAM gene is obtained by double enzyme digestion of the plasmid pMD18-T-rSLAM respectively through Xho I and EcoR I.

The Pcvv, IRES, EGFP and SV40pA are obtained by double enzyme digestion of pIRES2-EGFP vector by Xho I and EcoR I.

A Vero cell line, wherein the eukaryotic expression recombinant plasmid gene is cloned in Vero cell genome, and a canine distemper virus receptor raccoon SLAM (rSLAM) can be stably expressed on the cell surface.

The construction method of the Vero cell line can be as follows: designing 1 pair of primers, taking pMD18-T-rSLAM plasmid with cloned raccoon SLAM gene as a template, obtaining a Kozak sequence + raccoon SLAM +6 × his fusion gene by a PCR method, and constructing pIRES2-EGFP-rSLAmhis plasmid; the pIRES2-EGFP-rSLAMhis plasmid is transfected into Vero cells, and Vero cell lines which stably express the SLAM are screened by observing the expression of the EGFP and adopting G418 pressure culture clone.

The invention also provides application of the Vero cell line in rapid and sensitive separation of canine distemper virulent strains and research of canine distemper viruses.

The invention constructs and transfects eukaryotic expression recombinant plasmid pIRES2-EGFP-rSLAMHis (composed of a raccoon SLAM gene with the GeneBank accession number of EU678639, a fusion gene of a nucleotide sequence coding 6 histidines and a Kozak sequence, Pcvv, IRES, EGFP and SV40pA, wherein the 5 'end to the 3' end of the plasmid is Pcvv in sequence, and the fusion gene, IRES, EGFP and SV40pA are added to a Vero cell line, Vero cells of a canine heat virus receptor rSLAM with stable surface and high expression are obtained through cloning and screening, and then tissue sample isolation viruses containing the canine heat virus are inoculated, and the virus inoculation test result shows that the Vero cells after transfection of the rSLAM enhance the sensitivity to CDV and can effectively separate the canine heat virus strains.

The invention also provides a canine distemper virulent strain LN (10) f1 strain separated from the Vero cell line.

The invention provides a eukaryotic expression recombinant plasmid, a construction method thereof and a Vero cell line for stably expressing the plasmid. In order to enhance the translation efficiency of a target protein gene (rSLAM) and facilitate the detection of protein expression, Kozak sequences are respectively introduced at the upstream and the downstream of the rSLAM gene when the primers are designed. The invention introduces 6 histidine tags at the carboxyl terminals of the expressed protein, is convenient for applying the histidine tag monoclonal antibody (the monoclonal antibody is commercialized at home and abroad) to carry out semi-quantitative evaluation on the expression of the SLAM protein by Western blot and cell immunohistochemistry, and has important effect on the identification of constructing a stable expression cell line. The Kozak sequence can obviously improve the expression quantity of SLAM protein when in plasmid expression, is beneficial to improving the combination efficiency of CDV and the CDV, and improves the separation efficiency of expression cell line virus. According to the invention, a eukaryotic expression vector pIRES2-EGFP is adopted as a binary expression vector, an inserted target gene and EGFP are co-expressed under a CMV promoter, so that the expression can be monitored, the property of a target protein can be well maintained, a positive cell can be positioned by green fluorescent protein at the initial stage of cell screening, the monoclonal selection of an expression protein cell is carried out, and the success rate of screening is improved by combining G418 resistance screening. The pIRES2-EGFP-rSLAMh plasmid is transfected into Vero cells, when the transfected cells grow for 48 hours, the expression rate of the EGFP is higher after the pIRES2-EGFP-rSLAMh plasmid is transfected. After 10 serial screenings, EGFP was still expressed.

The invention successfully detects the expression of the SLAM fusion protein after transfection by aiming at the cell immunohistochemistry of 6 histidine tags, and proves that the sensitivity of the cells to CDV is really enhanced after the SLAM is transfected through a virus inoculation experiment of transient transfection Vero cells, and also shows that the fusion of 6 His tag proteins at the C' end of the SLAM does not lead the SLAM protein to lose the recognition and combination capability with CDV H protein. Provides reliable experimental basis for the feasibility of establishing stably transfected cells. After 10 generations of continuous screening of the transfected cells, the expression of SLAM fusion protein and the expression of EGFP protein can be detected in the cells of the immunohistochemical cells. The cell doubling time of the Vero cells and the V-p-rSLAMH cells can be calculated according to the drawn growth curves of the transfected cells (V-p-rSLAMH cells) and the untransfected Vero cells, the cell doubling time of the Vero cells is 16.69h at the shortest time, and the cell doubling time of the V-p-rSLAMH cells is more than 16.69h, so that the growth speed of the cells after the foreign proteins are expressed becomes slow, the metabolic burden of the cells is increased by the expression of the foreign proteins, the growth speed naturally becomes slow, and the V-p-rSLAMH cells are larger in appearance than the Vero cells. The method successfully establishes the Vero cell line for stably expressing the raccoon dog SLAM, and has important significance for the rapid separation of CDV and the establishment of a research platform.

V-p-rSLAMH cells and Vero cells are respectively used for inoculating 3 dead fox or racoon dog canine distemper suspected tissue samples, the V-p-rSLAMH cells can form visible typical CDV CPE in the first generation of inoculation for 36-48 h, and the Vero cells do not form CPE in the continuous blind 6 generations. V-p-rSLAMH cells were shown to be CDV sensitive compared to Vero cells. RT-PCR verifies that the separated 3 strains are CDV, and the separated 3 CDV H genes are subjected to clone sequencing and then are compared, so that the results show that the 3 strains CDV are Asia-1 genotype wild strains; 1 of them (designated LN (10) f1 strain)]After the V-p-rSLAMH cells are continuously transmitted for 4 generations, the toxicity of the animals is verified by an animal challenge experiment and is 4 multiplied by 10^2.39TCID₅₀The dose of each animal can lead the raccoon dog and the fox (each animal has 3 animals) of the offending group to die. The CDV separated by the V-p-rSLAMH cell is proved to maintain the virulent toxicity, and the lethality rate to the inoculated animal can reach 100 percent.

The invention establishes a Vero cell line for stably expressing the raccoon dog SLAM receptor, systematically evaluates the stability of the Vero cell line for expressing the raccoon dog SLAM receptor and the sensitivity to CDV, and proves that the cell line has stable target gene expression and is sensitive to CDV virus, and the CDV obtained by separation has stronger toxicity. The establishment of Vero cell line for stably expressing raccoon dog CDV SLAM provides a platform for the separation and research of CDV. In addition, in the process of developing a vaccine for preventing CD, the vaccine efficacy evaluation is indispensable to CDV virulent virus with stable toxicity, and the acquisition of the CDV virulent virus of LN (10) f1 strain provides an evaluation tool of high-efficiency vaccine, and has great significance for the epidemic prevention of CD.

Drawings

FIG. 1 shows the recovery of rSLAM target fragment from the gel after PCR; wherein, Lane 1 is 100bp DNA Laddermarker, Lane 2 is the target fragment amplified by PCR using the plasmid pMD18-T-rSLAM as a template, and the size is about 1100 bp.

FIG. 2 shows the results of enzyme digestion identification of the recombinant expression vector; wherein, Lane 1 is 100bp DNAladder Marker, Lane 2 is DL15000DNA Ladder Marker, Lane 3 is pIRES2-EGFP-rSLAMH, and the two bands with the size of about 1100bp and 5300bp are generated by double enzyme digestion.

FIG. 3 illustrates the determination of G418 working concentration; wherein, FIG. 3(a) is Vero cells without G418; FIG. 3(b) is Vero cells containing 200. mu.g/mL G418; FIG. 3(c) is Vero cells containing 600. mu.g/mL G418; FIG. 3(d) is Vero cells containing 800. mu.g/mL G418.

FIG. 4 shows EGFP expression 48h after transient transfection of Vero cells with recombinant plasmids; wherein FIG. 4(a) is an untransfected Vero cell; FIG. 4(b) pIRES2-EGFP transfected Vero cells; FIG. 4(c) is pIRES2-EGFP-rSLAMH transfected Vero cells.

FIG. 5 shows CDV-detoxified 60h cytopathic effect (CPE) after transient transfection of Vero cells with recombinant plasmids; wherein FIG. 5(a) is an untransfected Vero cell; FIG. 5(b) pIRES2-EGFP transfected Vero cells; FIG. 5(c) is pIRES2-EGFP-rSLAMH transfected Vero (arrows indicate cytopathic location).

FIG. 6 shows that Vero cells transfected for 48h are replaced by 10% FBS screening medium with the G418 content of 800. mu.g/mL, screened and cultured for about 10 days, then transferred to a 100mm cell culture dish according to the ratio of 1: 40 after being digested by pancreatin, and then screened by continuously adding the screening medium to obtain transfected Vero cell clusters.

FIG. 7 shows the results of a cytoimmunohistochemical assay; among them, FIG. 7(a) is Vero cells, FIG. 7(b) is Vero cells transfected with empty vector, and FIG. 7(c) is transfected recombinant plasmid Vero cells (showing SLAmh protein expression).

FIG. 8 shows growth curves of transfected cells versus untransfected cells; wherein,

showing a Vero cell, a cell line,vero cells (V-p-rSLAMh cells) after pIRES2-EGFP-rSLAMh transfection are shown.

FIG. 9 shows cytopathic after vaccination; among them, FIG. 9(a) shows Vero cells after inoculation, FIG. 9(b) shows V-p-rSLAMH cells inoculated with JL (10) r1 strain CDV 48h, FIG. 9(c) shows V-p-rSLAMH cells inoculated with HeB (10) f1 strain CDV 48h, and FIG. 9(d) shows V-p-rSLAMH cells inoculated with LN (10) f1 strain CDV 24 h.

FIG. 10 shows the results of RT-PCR amplification of CDV isolated from V-p-rSLAMH cells. Among them, lane 1 is 100bp DNAladder Marker, lane 2 is JL (10) r1 strain, lane 3 is HeB (10) f1 strain, lane 4 is LN (10) f1 strain, and lane 5 is a positive control.

Fig. 11 shows the body temperature of the challenge and control raccoon dogs; wherein,

the body temperature of the No.1 raccoon dog is shown,

showing the body temperature of the No.2 raccoon dog,

showing the body temperature of the No.3 raccoon dog,

the body temperature of the raccoon dog is shown.

Figure 12 shows body temperatures of challenge and control foxes; wherein,

showing the body temperature of the fox No.1,

showing the body temperature of the fox No.2,

showing the body temperature of the fox No.3,body temperature of control foxes is shown.

FIG. 13 shows eukaryotic expression vector pIRES2-EGFP map.

FIG. 14 shows a eukaryotic expression vector pIRES2-EGFP circular map.

FIG. 15 shows a map of eukaryotic expression recombinant plasmids provided by the present invention.

Detailed Description

The invention discloses a eukaryotic expression recombinant plasmid, a construction method thereof and a Vero cell line for stably expressing the plasmid, and can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Eukaryotic expression vector pIRES2-EGFP was purchased from Clontech, USA; the vectors for restriction enzymes XhoI and EcoR I were purchased from Takara Shuzo (Dalian) Co., Ltd; the Vero cells are preserved in a preventive veterinary research laboratory of a specialty institute of Chinese academy of agricultural sciences; the Midiprep High-pure plasmid extraction kit was purchased from Invitrogen; DMEM medium was purchased from Gibco BRL; g418 was purchased from Promega; transfection reagent

HD transformation Reagent from Roche;

Monoclonal Antibody is available from Novagen corporation; the cellular immunohistochemistry kit SP kit (Mouse) is purchased from Beijing Boaosen biotechnology, Inc.; trizol was purchased from Invitrogen, USA; inverted fluorescence microscope (DMI3000B) from come, germany; the suspected canine distemper fox tissue sample is preserved in a preventive veterinary research laboratory of a specialty institute of Chinese academy of agricultural sciences.

The invention is further illustrated by the following examples:

EXAMPLE 1 construction of eukaryotic expression recombinant plasmid pIRES2-EGFP-rSLAmh

PCR amplification and gene cloning: taking the plasmid pMD18-T-rSLAM as a template, and

P₁5’-CCG

ATGGATTCCAGGGGCTTCCTC-3' is upstream primer, and P is₂5’-CCGTCAGTGATGGTGATGGTGATGGCTCTCTGGGAACGTCAC-3' as downstream primer (box is restriction enzyme cutting site, bold is Kozak sequence, underline is histidine label gene), PCR amplification is carried out to obtain target gene, reaction system is: 1. mu.L (0.2 ng/. mu.L) of DNA template, 1. mu.L (20 pmol/. mu.L) of each of upstream and downstream primers, 0.3. mu.L (5U/. mu.L) of Ex taq enzyme, 2. mu.L of dNTP (2.5 mmol/. mu.L each), 2. mu.L of 10 XPCR Buffer, 2.5. mu.L of ddH₂O make up to 25. mu.L.

The amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61.8 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; total extension at 72 ℃ for 10 min; storing at 4 deg.C for 10 min. The PCR product was recovered by 1% agarose gel, ligated into pMD18-T vector, and subjected to restriction enzyme identification.

The diluted plasmid pMD18-T-rSLAM is used as a template, a target fragment is amplified by PCR, the obtained target fragment is recovered and purified by 1 percent agarose gel, a band with the expected size of about 1100bp can be seen under an ultraviolet transilluminator after electrophoresis for 45min under the voltage of 100V, and an image is collected by an ImageQuant 3000 gel imaging system (GE company) as shown in figure 1.

Construction of eukaryotic expression vectors: plasmid pMD18-T-rSLAmh was double digested with XhoI and EcoRI, and the gel recovered fragment of interest was inserted into the vector fragment recovered from the XhoI and EcoRI double digested pIRES2-EGFP gel under the action of T4 ligase. The ligated vector was transformed into the cloned strain DH5 α, and spread on LB plates resistant to kanamycin (30. mu.g/mL); after 24h of culture, aseptically picking the monoclonal strains in 3-4 mL test tubes of liquid LB with kanamycin resistance (30 mug/mL), putting the test tubes into a constant temperature shaking incubator (37 ℃, 200r/min) overnight; plasmid extraction kit (Hangzhou Bori) is used for extracting plasmid according to the instruction.

Enzyme digestion identification: the pIRES 2-EGFP-rSLAMHh recombinant plasmid is subjected to double enzyme digestion to generate two bands with the sizes of about 1100bp and 5300 bp. The result is shown in FIG. 2, which shows that the eukaryotic expression plasmid is successfully constructed, and pIRES2-EGFP-rSLAMh is connected with the target fragment.

The constructed eukaryotic expression plasmid is sent to a biological company for sequencing, and the sequence has a nucleotide sequence shown as SEQ No. 1.

Example 2 establishment of Vero cell line stably expressing pIRES2-EGFP-rSLAMH

Determination of the optimum concentration of G418: after the recovered Vero cells are propagated for 2-3 generations by using 10% FBS DMEM, the cells to be passaged are digested by pancreatin, and the ratio of each hole is 1 multiplied by 10⁴The number of cells/well was plated in 96-well plates, the plates were plated on day 2 and the medium was aspirated, the cells were washed once with PBS and the selection medium was added. The concentration of G418 was 0, 200, 300, 400, 500, 600, 700, 800, 900, 1000. mu.g/mL in this order, 3 replicates were used for each gradient, the selection medium was changed every 3 days or so depending on the color of the medium, the culture was continued for 10 days, the cell death was observed every day, and the lowest G418 concentration at which all cells were dead on day 10 was used as the optimum selection concentration. Vero cells with different concentrations of G418 are added, and a small amount of cells are dead in the screening group on the 2 nd day; at 8 days of screening, 95% of the cells died in the 800. mu.g/mL group, 99% of the cells died in the 900. mu.g/mL group, and 100% of the cells died in the 1000. mu.g/mL group; screening for 10 days (see figure)3) The 200 ug/mL group had 40% cell death, the 600 ug/mL group had 90% cell death, and the cells in the groups above 800 ug/mL all died, and the optimal screening concentration for Vero cell G418 was determined to be 800 ug/mL.

Preparation of high purity plasmid for transfection: the pIRES2-EGFP and pIRES2-EGFP-rSLAMh strains prepared in example 1 were inoculated into 100mL of Kan-resistant LB medium at 37 ℃ and 200r/min, respectively, overnight. The double expression vector is purified and extracted by using a Midiprep High-pure plasmid extraction kit according to the operation instruction. Plasmids were extracted according to the procedure described in the specification.

Transient transfection of cells: the well-grown cells were cultured at 1X 10 per well⁵The density of each cell was plated on 6-well plates (6 wells were set as untransfected plasmid group, pIRES2-EGFP empty vector group, pIRES2-EGFP-rSLAmh group, and Vero cells transfected with empty vector and recombinant plasmid were named V-p and V-p-rSLAmh, respectively) at 37 ℃ with 5% CO₂After overnight incubation in the incubator, cell transfection was performed when the cell confluence was about 80% according to the following procedure: before transfection, serum-free DMEM, purified plasmids and transfection reagent are placed in a room temperature environment; adding 100 mu L of DMEM without serum and antibiotics into a 1.5mL sterile and siliconized EP tube, then adding 2 mu g of plasmid, fully mixing, using a pipettor with a siliconized pipette tip, hanging and dropping 9 mu L of HD transfection reagent (mixing before adding transfection reagent), reversing and mixing; incubation for 15min at room temperature to allow formation of transfection complexes; dropwise adding the transfection complex in the EP tube into a corresponding 6-well plate, and slightly rotating the 6-well plate in the dropwise adding process to ensure that the transfection complex is uniformly distributed on the cell surface; 37 ℃ and 5% CO₂After 48h incubation in the incubator, images were collected under a fluorescence microscope. As shown in FIGS. 4(a) to 4(c), it was found that the expression rate of EGFP was high after pIRES2-EGFP-rSLAmh plasmid transfection. Obvious CPE can be seen 60h after CDV inoculation of the recombinant plasmid group, no CPE is seen in Vero of the untransfected group and the transfection empty plasmid group, and the results are shown in fig. 5(a) to fig. 5(c), so that rSLAM expression after transfection can obviously improve the susceptibility of CDV to cells.

Validation of SLAM expression after transient transfection:

transfected cell plates were incubated at 37 ℃ with 5% CO₂After culturing in an incubator for 48h, observing by a fluorescence microscope, selecting a plate of cells, inoculating 200 mu L of treated canine distemper fox organ poison LN (10) f1 strain (DMEM cell maintenance liquid which is changed by 2% FBS after adsorbing 1 h) into each hole, and observing whether typical CDV infected cytopathic condition occurs or not every day after inoculation. The process of treating the visceral virus is as follows:

placing lung tissue with serious pathological changes into a mortar, mincing the tissue with small scissors, adding a small amount of quartz sand and PBS, and quickly grinding on ice; the ratio of the pathological material to PBS is about 1: 5(g/mL), and the pathological material is put into a refrigerator with the temperature of 80 ℃ below zero for freezing and thawing once; centrifuging at 4 deg.C and 3000r/min for 30min, collecting supernatant, and filtering with 0.22 μm filter for sterilization; after the culture solution of the cells was aspirated before inoculation, the cells were washed 3 times with PBS, 200. mu.L of each well was inoculated and adsorbed for 1 hour, and then cell maintenance solution of DMEM containing 2% FBS was added.

Clonal screening of stably expressing SLAM cells: stable transfected cell clone selection was performed using a combination of G418 pressure selection and cell cloning loop. Transfected cell plates were incubated at 37 ℃ with 5% CO₂After 3 days of culture in the incubator, 10% FBS DMEM containing G418 of 800. mu.g/mL was replaced with the selection medium, the growth state of the cells was observed every day, the selection medium was replaced every 3 days until more than 99% of the cells in the control wells were dead, the cells were digested with 0.25% pancreatin, and the cells were transferred to a 100mm large plate at a dilution of 1: 30. Culturing by using a 10% DMEM screening culture medium containing 800 mu G/mL G418, changing a new culture medium every 3 days, continuously culturing for about 10 days, observing the protein expression condition under a fluorescence microscope, marking the position with green fluorescence and a cell cluster by using a marker pen, digesting the cell at the position by using a cloning ring, transferring the cell to a large plate with the thickness of 100mm, and repeating the operation for 3-4 times to obtain the pure cell with stable expression. Cells were frozen every 5 passages during passage of cells.

And (3) changing the screening culture medium of 10% FBS with the G418 content of 800 mu G/mL for 48h of the Vero cells for transfection, changing the screening culture medium once every 3 days, carrying out screening culture for about 10 days, completely killing the normal control cells, transferring the cells to a cell culture dish with the diameter of 100mm according to the ratio of 1: 40 after the cells are digested by pancreatin, and continuously adding the screening culture medium to screen the transfected Vero cells. Under the microscope, cell cluster formation was observed (as shown in FIG. 6). And (3) observing by a fluorescence microscope, digesting the marker which is formed by the cell cluster and has a large amount of EGFP expression by using a marker pen by using a cloning ring, screening and culturing in a 6-well plate, and continuously culturing for 3-4 times to obtain a relatively pure cloning cell line. The obtained cell line is marked to be frozen for several times, and after 10 generations of V-p and V-p-rSLAMH groups are continuously screened, the EGFP is still stably expressed.

Verification of the expression of SLAM protein in a stably expressing cell line: stably transfected cells expressing different receptors were plated in 6-well plates with normal cells and cells with empty vector. The expression of stable cell line proteins was verified by a cellular immunohistochemistry technique. After the cells are cultured for 3-4 days, incubating for 30min at 37 ℃ with PBS; fixing with 2mL of methanol at 4 ℃ for 4 h; washing with PBS for 3min × 3 times, and washing with methanol for 3min × 3 times; after drying at room temperature, washing with 1% Tween-20 PBS for 3min × 3 times; 3% H₂O₂Incubating for 10-15 min by using the deionized water to eliminate the activity of endogenous peroxidase; incubating for 10-15 min at room temperature with a confining liquid, and pouring off without washing; dropwise adding histidine-primary antibody diluted by 1: 1200, and incubating at 37 ℃ for 2-3 h or overnight at 4 ℃; washing with PBS for 3min × 3 times; adding biotin-labeled goat anti-mouse IgG dropwise, and incubating for 10-15 min at room temperature or 37 ℃; washing with PBS for 3min × 3 times; dripping horseradish enzyme-labeled ovalbumin, and incubating for 10-15 min at room temperature or 37 ℃; washing with PBS for 3min × 3 times; developing the DAB color developing agent for 5-15 min, and fully washing with PBS; counterstaining with hematoxylin; PBS was washed clean and observed under microscope.

Screening 5 generation transfected cells and untransfected Vero cells, spreading on 6-well plate, culturing Vero cells in normal DMEM medium, culturing transfected cells in screening medium at 37 deg.C and 5% CO₂After 3 days in the incubator, the expression of the target protein was detected using the cell immunohistochemistry kit SP kit (Mouse), and as a result, the formation of a brown yellow precipitate was detected in both cells of the transfected group, demonstrating that the expression of SLAM was observed, as shown in FIG. 7(c), while no brown yellow precipitate was formed in the Vero cell group and the empty vector group, as shown in FIG. 7(a) and FIG. 7 (b).

Drawing a cell growth curve: recovering the cells expressing the canine distemper virus receptor and the normal Vero cells which are stably screened for 2 generations, after 2 generations of cells are recovered, paving a 24-hole plate, counting the cells every 24 hours or 12 hours, and drawing a cell growth curve according to the counting result, wherein the cell growth curve is shown in figure 8. Each group of cells grows vigorously within 72-108 h; vero cells began to decrease after 108 h.

Example 3

Comparison of sensitivity of cell lines to CDV: when Vero and V-p-rSLAMH cells had good growth status, 3 wells of a six-well plate (Nunc, Denmark) were plated with two types of cells, Vero cells were grown in 10% FBS-containing DMEM, and V-p-rSLAMH cells were grown in 800. mu.g/mL G418 at 37 ℃ with 5% CO₂After culturing in the incubator of (1) for 48 hours, the virus was aspirated, and the maintenance solution of V-p-rSLAMH cells also contained 800. mu.g/mL of G418, and the pathological changes of the cells were observed every 12 hours. The 3 wild strains inoculated are respectively the organ toxicity of Hebei fox in 2010 [ HeB (10) f1](ii) a Organ poison of Jilin Jitai racoon dog in 2010 (JL (10) r 1)](ii) a 2010 Liangning Jinzhou fox viscera toxin [ LN (10) f1)]. The 3 canine distemper samples are positive to canine distemper virus nucleic acid through RT-PCR detection. Cells without lesions were blind-passed, and those with no CPE were considered negative in the 6 th passage.

Isolation of 3 wild strains by two cells:

V-p-rSLAmh cells: and after the first generation of virus inoculation is carried out for 36-48 h, 3 wild strains have obvious cell fusion CPE. LN (10) f1 showed significant CPE at 36h, while the remaining 2 strains showed significant CPE at 48h, see FIG. 9(b), FIG. 9(c), FIG. 9 (d).

Vero cells: no CPE was seen in the 3 wild strains in the first generation, and no CPE was seen in the blind 6 generations of cells thereafter, which was considered as negative for virus isolation. See FIG. 9(a)

RT-PCR validation and H gene sequence analysis of the isolated virus:

after freezing and thawing 3 CDV strains separated from V-p-rSLAMH cells, respectively extracting virus RNA, reversely transcribing cDNA, and verifying by RT-PCR, the 3 separated virus strains are all CDV, as shown in figure 10.

Design and synthesis of primers: according to the CDV N and H gene sequences published on Genbank, a pair of detection primers are respectively designed in the conserved regions, the amplification fragment degree is 430bp, and an upstream primer (P1): 5 '-ACCAGACAAAGTTGGCTAWGGAT-3', downstream primer (P2): 5'-ATGCTTGGTATTACCTCTACTAACTTG-3', respectively; a pair of amplification primers, the amplification length is 1879bp, and an upstream primer (P3): 5'-TTAGGGCTCAGGTAGTCCA-3', downstream primer (P4): 5 '-CTAAGKCCAATTGARATGTGT-3', the designed primer is synthesized by Dalibao bioengineering GmbH.

Extraction of viral RNA, synthesis of cDNA and PCR amplification: CDV viral RNA was extracted by Trizol method as described above, followed by reverse transcription to synthesize cDNA, the reverse transcription system was as follows:

using cDNA as a template to amplify a specific fragment by using PCR, wherein the PCR reaction system is as follows: mu.L of DNA template, 1. mu.L of each of the upstream and downstream primers (20pmol/L), 0.3. mu.L of Ex Taq DNA polymerase, 2.5. mu.L of 10 XPCR buffer, 2. mu.L of dNTPs (2.5 mmol/L each), and 25. mu.L of ddH 2O. PCR amplification procedure: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 40s (2min), extension at 72 ℃ for 40s (2min), 35 cycles; finally, extension is carried out for 10min at 72 ℃. The PCR product was identified in 1% agarose. After recovery of the PCR amplification product gel, it was cloned into pMD18-T vector, respectively. After enzyme digestion identification, 3 positive clones of each fragment were selected and sent to Dalianbao bio-corporation for sequencing to determine a consensus sequence.

CDV H sequence analysis: and (3) sequencing the correct H gene, comparing and analyzing gene sequences of other CDV strains registered by GenBank by using DNASAR software MegAlign/Clustal W, constructing a phylogenetic tree, and predicting potential N-linked glycosylation sites of the H protein by using NetNGlyc 1.0Server software.

LN (10) f1 and [ JL (10) r1 isolated from V-p-rSLAMHh cells]And [ HeB (10)f1]The comparison of the H gene sequence of the strain CDV with other strain sequences shows that 3 strains of viruses are all CDV and belong to Asia-1 genotype wild strains. The number of potential N-linked glycosylation sites of the H gene is 8-9 wild strains, and 4 (Onderstepopoort) or 7 (CDV) vaccine strains₃) The 309-311N-linked glycosylation sites are the specific glycosylation sites of the wild strains, and the amplified 3 CDV H genes contain 9 glycosylation sites including 309-311 sites, so that the characteristics of pathogenic wild strains (virulent strains) are met. Wherein the nucleotide sequence of the LN (10) f1 strain H gene has only 91.2 percent of homology with the vaccine strain Onderstepopoort (AF305419) and CDV3, and has 98.4 percent of homology with the Asia-1 genotype HBF-1 wild strain (EU 325721). It was confirmed that the isolated LN (10) f1 strain CDV was a wild strain of Asia-1 genotype.

Determination of the CDV virus content: V-p-rSLAmh cells in good growth status were selected, digested with 0.25% trypsin, and then grown in DMEM medium containing 10% FBS (containing 800. mu.g/mL of G418) at a cell concentration of 3X 10⁵and/mL. Add 100. mu.L per well to a 96-well plate using a line gun. Place the plates in 5% CO₂Culturing at 37 deg.C for 12-18h in incubator to allow cell to grow into monolayer. Serially diluting the virus to be detected by 10 times with DMEM maintenance solution containing 2% FBS to make the concentration of the virus solution be 10^-1～10^-6. The growth medium was discarded, and the cells in each well were washed 2 times with PBS, and then 10 times from the virus solution^-2Starting dilution, adding 100 μ L of virus solution with different dilutions into each well, repeating each dilution with 8 wells, making two rows as positive control, making one row as normal control without virus solution, adsorbing at 37 deg.C for 1 hr, adding 100 μ L of maintenance solution (containing 800 μ G/mL G418) into each well, shaking gently, and adding 5% CO at 37 deg.C₂And (5) carrying out static culture in an incubator. Observing cytopathic effect of each well day by day for 5 days, and calculating TCID by Reed-Munch method₅₀。

LN (10) f1 strain animal challenge experiment: continuously propagating the separated LN (10) f1 strain in V-p-rSLAMHh cells for 4 generations, freezing and thawing the cells for 2 times for foxes (raccoon dogs) to counteract the virus, and determining the virus content before counteracting the virus. Selecting 5 foxes (raccoon dogs) with negative canine distemper virus neutralizing antibody (less than 1: 4), and 3 foxes (raccoon dogs) with positive canine distemper virus neutralizing antibodyTest group, 2 only as control group. Adopts a mode of subcutaneous multi-point injection to counteract poison of each fox (raccoon dog) in an experimental group, and the dose of counteracting poison is 4 multiplied by 10^2.39TCID₅₀Control group was injected subcutaneously with the same dose of DMEM nutrient solution at multiple points. The body temperature of each fox (raccoon dog) was measured every morning and observed to record the status of each fox (raccoon dog).

The raccoon dog with the function of counteracting toxic pathogen has the clinical symptoms:

the body temperature of the toxicity counteracting group starts to rise to more than 39.5 ℃ from 4 days, reaches the highest value (more than 41 ℃) in 5-9 days, then the body temperatures of the

raccoon dogs

2 and 3 fall, the raccoon dog # 2 starts to rise again after opening for 11 days, and the toxicity counteracting raccoon dogs die in 11-13 days as shown in fig. 11. The raccoon dog in the control group is normal in body temperature (below 39.5 ℃) in the whole test process.

The clinical symptoms of the toxin-counteracting fox are as follows:

the disease course of the fox attacking toxin is long, the body temperature of the fox attacking group starts to rise to more than 40 ℃ from 4 days, and the CDV of the anus swab of the No.3 fox on the 10 th day is positive; on 12-13 days, the attacking group begins to have appetite reduction, conjunctivitis, bloody stool, dry nose and serious thickening inflammation of foot pads; the 3 toxin-attacking foxes died in 18 th to 22 th days, and the control group had good appetite and mental status and normal body temperature (below 39.5 ℃), as shown in fig. 12.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A eukaryotic expression recombinant plasmid having the sequence of SEQ ID NO: 1.

2. The method for preparing the plasmid of claim 1, comprising:

step 1: taking pMD18-T-rSLAM plasmid as a template, and amplifying in the presence of an upstream primer and a downstream primer to obtain the fusion gene, wherein the sequence of the upstream primer is shown as SEQ No.2, and the sequence of the downstream primer is shown as SEQ No. 3;

step 2: pIRES2-EGFP vector is subjected to enzyme digestion to obtain Pcvm, IRES, EGFP and SV40 pA;

and step 3: and (3) taking the fusion gene for enzyme digestion, connecting the fusion gene with Pmv, the IRES, the EGFP and the SV40pA in the step 2, transforming a recipient bacterium, and identifying a positive clone to obtain the plasmid.

3. The method of claim 2, wherein the enzyme is a Xho I and EcoR I double enzyme.

4. A Vero cell line, characterized in that after the Vero cell is transfected with the eukaryotic expression recombinant plasmid of claim 1, the cell line with canine distemper virus receptor raccoon SLAM stably expressed on the cell surface is obtained through cloning and screening.

5. The Vero cell line of claim 4 for isolation of a virulent strain of canine distemper virus and its use in studies of canine distemper viruses.