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Recombinant Vero cell line capable of stably expressing porcine delta coronavirus-N protein and application of recombinant Vero cell line

A coronavirus, stable expression technology, applied in the field of biotechnology detection, can solve the problem of lack of effective means for epidemic monitoring and achieve good specificity

Inactive Publication Date: 2019-06-21
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective antiviral treatment and vaccine for the infection of this virus, and there is also a lack of effective means for its epidemic monitoring

Method used

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  • Recombinant Vero cell line capable of stably expressing porcine delta coronavirus-N protein and application of recombinant Vero cell line
  • Recombinant Vero cell line capable of stably expressing porcine delta coronavirus-N protein and application of recombinant Vero cell line
  • Recombinant Vero cell line capable of stably expressing porcine delta coronavirus-N protein and application of recombinant Vero cell line

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1 Construction of recombinant expression vector pLVX-EF1α-IRES-Puro-PDCoV-N

[0029] The target sequence was amplified by PCR. According to the N gene sequence of PDCoVCHN / Tianjin / 2016 strain determined by our laboratory (Gen Bank accession number: KY065120), the terminal stop codon TAG was removed, and the primer design software PrimerPremier 5 was used to design primers for PCR amplification. For the convenience of cloning, BamH I and Xho I restriction endonuclease recognition sites were added to both ends of the gene fragment; for the convenience of subsequent identification, a Flag tag sequence was added at the 3' end of the gene. The designed PCR primers are:

[0030] PDCOV-N-F (SEQ ID NO.1): 5'-GAA CTC GAG ATC ATG GCT GCA CCA GTA GTC-3'

[0031] PDCOV-N-R (SEQ ID NO.2): 5'-GTT GGA TCC CTA CTT GTC GTC ATC GTC TTTGTAGTC CGC TGC TGA TTCTT-3'

[0032] The added Flag tag sequence is: 5'-CTT GTC GTC ATC GTC TTT GTA GTC-3' (SEQ ID NO.3)

[0033] Construction o...

Embodiment 2

[0034] The establishment of embodiment 2 recombinant cell lines Vero / PDCoV-N

[0035] The recombinant cell line Vero / PDCoV-N that detects PDCoV antibody described in the present invention can be obtained by the following steps:

[0036] Packaging of lentivirus: inoculate the constructed recombinant bacteria Stbl2 / pLVX-EF1α-IRES-Puro-PDCoV-N in the liquid LB medium supplemented with ampicillin at a ratio of 1:100, and culture with vigorous shaking at 250r / min for 12~ After 16 hours, use the endotoxin extraction plasmid kit to extract the pLVX-EF1α-IRES-Puro-PDCoV-N plasmid, and combine the extracted pLVX-EF1α-IRES-Puro-PDCoV-N lentiviral expression plasmid with two auxiliary plasmids pMD2.G and psPAX2 use 3000 transfection reagents were used to transfect 293T cells for virus packaging (the total mass of plasmids was 15 μg, and the ratio of plasmids was 7:2:1), and the supernatant was collected after 48h to 72h, and the supernatant was collected again at intervals of 48h to 72...

Embodiment 3

[0041] Application of embodiment 3 cell line Vero / PDCoV-N

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Abstract

The invention belongs to the field of biotechnology detection and particularly relates to a recombinant cell line capable of stably expressing porcine delta coronavirus-N protein and application of the recombinant cell line. The cell line comprises a porcine delta coronavirus-N protein gene and a resistance screening gene. The obtained recombinant cell line capable of stably expressing PDCoV-N protein and with puromycin resistance can be used for detecting PDCoV specific antibodies, and the fact that a swine herd which is not immunized by vaccines is infected by wild viruses can be determinedwhen specific antibody positive reaction is detected.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a recombinant cell line stably expressing porcine delta coronavirus N protein and application thereof. Background technique [0002] Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus that can cause diarrhea in pigs, especially newborn piglets, and its clinical manifestations are similar to those of known porcine transmissible gastroenteritis and porcine epidemic diarrhea Very similar, easy to confuse, infection can lead to higher morbidity and mortality, and it is one of the important reasons for the death of newborn piglets in many countries and regions in recent years. PDCoV was first discovered by Hong Kong scholars in 2012 (WooPC, et al. virology, 2012,86(7):3995-4008.), the first large-scale epidemic occurred in the United States in 2014 (Li G, et al.Full-Length GenomeSequence of Porcine Deltacoronavirus Strain USA / IA / 2014 / 8734.Genomeannou...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/50C12Q1/70
Inventor 张小荣钱炳旭吴艳涛陈杨郭梦娇张成成曹永忠
Owner YANGZHOU UNIV
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