摘要
蓝舌病病毒(Bluetounge virus,BTV)非结构蛋白4(Non-structural protein4,NS4)拮抗干扰素的产生,在BTV逃避宿主天然免疫的过程中发挥重要作用。利用PCR扩增得到NS4基因,与空载体pET-32a双酶切后连接,构建pET-32a-NS4原核表达载体;将pET-32a-NS4转化至E.coli BL21感受态细胞,以IPTG诱导表达;利用SDS-PAGE确定IPTG的最佳诱导浓度、时间与重组蛋白的表达形式;利用Western blot分析重组蛋白的表达情况。结果表明,成功构建pET-32a-NS4原核表达载体;IPTG最佳诱导浓度为0.8mmol/L,时间为6h,蛋白主要以包涵体形式表达;Western blot在27ku处可见目的条带,与预期相符。本研究成功构建pET-32a-NS4原核表达载体,实现NS4蛋白的大量、高效表达,为进一步探究NS4蛋白在BTV拮抗宿主天然免疫中的作用机制提供材料。
The non-structural protein 4(NS4)of Bluetounge virus(BTV)antagonizes the productionof interferon,which plays an important role in the escape of BTV from host innate immunity,In thisstudy,NS4 gene was amplified by PCR and ligated with pET-32a to construct a prokaryotic expressionvector pET-32a-NS4;pET-32a-NS4 was transformed into E.coli BL21 cells and induced by IPTG;SDS-PAGE was used to determine the optimal concentration and time of IPTG,and the expressionform of the recombinant protein was analyzed by Western blot.The results showed that pET-32a-NS4was successfully constructed,the optimal condition of IPTG was 0.8 mmol/L for 6 h;Western blotshowed the target band at 27 ku and the protein was mainly expressed in the form of inclusion body.In conclusion,pET-32a-NS4 is successfully constructed to achieve a large number of highly efficientexpression of NS4 protein.This study will provide materials for further exploration of the mechanismsof NS4 protein in BTV antagonism against host innate immunity.
作者
马骥
朱文青
林俊泓
易华山
胡庭俊
MA Ji;ZHU Wenqing;LIN Junhong;YI Huashan;HU Tingjun(College of Animal Science and Technology,Guangxi University,Nanning 530005,China;College of Veterinary Medicine,Southwest University,Rongchang Chongqing 402460,China)
出处
《西北农业学报》
CAS
CSCD
北大核心
2022年第9期1168-1173,共6页
Acta Agriculturae Boreali-occidentalis Sinica
基金
重庆市基础研究与前沿探索专项(cstc2018jcyjAX0615)。
关键词
蓝舌病病毒
NS4基因
载体构建
原核表达
Bluetounge virus
NS4 gene
Vector construction
Prokaryotic expression
