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

摘要/Abstract

摘要： 为探究猪痘病毒作为载体表达猪圆环病毒2型（PCV2）-Cap蛋白，插入外源基因P28-ORF2的拷贝数与PCV2-Cap蛋白表达量的关系。本试验以猪痘病毒为载体，通过双酶切连接及无缝克隆方法构建重组质粒，并纯化到了8株含不同拷贝数（1~8拷贝） P28-ORF2的重组猪痘病毒，基于荧光斑大小、电镜观察病毒粒子及Western blotting结果进行判断。纯化到的8株重组病毒的荧光斑直径为317.41~384.96 μm，大小无明显差异。重组猪痘病毒粒子形态与亲本病毒一致。Western blotting结果为插入1拷贝P28-ORF2的重组蛋白表达量最低；随着P28-ORF2拷贝数的增加，PCV2-Cap表达量也随之增加；至插入4拷贝P28-ORF2时，PCV2-Cap表达量达到峰值；随后减少。8株重组猪痘病毒荧光斑大小无明显差异，表明猪痘病毒基因组中插入不同拷贝数P28-ORF2对重组病毒在PK15细胞内的增殖无影响。重组猪痘病毒粒子的形态未发生变化说明多拷贝外源基因的插入并未对猪痘病毒的结构造成影响。本研究结果表明，猪痘病毒为载体表达外源蛋白时，单启动子启动多拷贝外源序列能明显提高外源蛋白的表达量，插入4拷贝的外源序列为较合适的拷贝数。

关键词: 重组猪痘病毒; 猪圆环病毒2型; 拷贝数

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

中图分类号:

S858.28

高映雪, 薛瑞, 林敏, 胡换仪, 刘昌锦, 万文忠, 罗锋, 邓舜洲. 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[J]. 中国畜牧兽医, 2021, 48(3): 818-828.

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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