表达猪圆环2型的多拷贝P28-Cap重组猪痘病毒的构建及表达量分析

doi:10.16431/j.cnki.1671-7236.2021.03.005

Abstract

Abstract: The purpose of this study was to explore the relationship between the copy number of P28-ORF2 inserted into the genome of PCV2-Cap and the expression of PCV2-Cap protein when Swine pox virus vector was used to express PCV2-Cap protein.In this experiment,using Swine pox virus as vector,8 strains of recombinant Swine pox virus with different copy number (1-8 copies) of P28-ORF2 were constructed by double enzyme digestion and in-fusion cloning method.8 strains of recombinant Swine pox virus with different copy number (1-8 copies) were purified.The virion particles were observed by electron microscope and the results of Western blotting were judged based on the size of fluorescent spot and electron microscope.The fluorescent spot diameter of the 8 purified recombinant virus was about 317.41-384.96 μm,and there was no significant difference in the size of the fluorescent spots.The morphology of the recombinant Swine pox virus was the same as that of the parent virus.The results of Western blotting showed that the expression of recombinant protein with 1 copy of P28-ORF2 was the lowest,and the expression of PCV2-Cap increased with the increase of P28-ORF2 copy number.When 4 copies of P28-ORF2 were inserted,the expression of PCV2-Cap reached the peak,and then decreased.There was no significant difference in the fluorescent spot size of 8 strains of recombinant Swine pox virus.The insertion of different copy numbers of P28-ORF2 into the genome of Swine pox virus had no effect on the proliferation of recombinant virus in PK15 cells.The morphology of recombinant Swine pox virus particles did not change,indicating that the insertion of multiple copies of foreign genes did not affect the structure of Swine pox virus.The results showed that when Swine pox virus was used as vector to express foreign protein,single promoter initiation of multi copy foreign sequence could significantly increase the expression of foreign protein,and the foreign sequence inserted 4 copies was a suitable copy number.

Key words: recombinant Swine pox virus; Porcine circovirus type 2; copy number

CLC Number:

S858.28

GAO Yingxue, XUE Rui, LIN Min, HU Huanyi, LIU Changjin, WAN Wenzhong, LUO Feng, DENG Shunzhou. Construction and Expression Analysis of Multicopies P28-Cap Recombinant Swine Pox Virus Expressing Porcine Circovirus Type 2[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 818-828.

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Construction and Expression Analysis of Multicopies P28-Cap Recombinant Swine Pox Virus Expressing Porcine Circovirus Type 2

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