摘要
为研究STAT1基因在ST细胞上的表达情况,笔者参考NCBI中多个物种的STAT1基因,把比对后显示序列保守的区域进行引物设计并进行合成。提取ST细胞的RNA,通过试剂盒进行反转录获得目的cDNA,再进行聚合酶链式反应(PCR),放大扩增猪的全长STAT1基因片段,对目的基因进行克隆、序列比对以及遗传进化分析。用内切酶Xhol和BamHI对测序无误的重组质粒pEGFP-STAT1进行双酶切,分离、纯化,与相同方法处理后的pEGFP-N1载体在适宜环境下进行体外连接,构建包含目的基因的重组质粒,进行双酶切、序列测定,之后再转染至ST细胞,运用West ern bl ot进行目的基因的蛋白检测来了解其表达情况。同等环境下猪传染性胃肠炎病毒(TGEV)感染ST细胞,qPCR检测不同接毒时间下ST细胞中STAT1的表达情况。结果表明,猪STAT1基因大小为2.274 kp;构建的重组真核表达质粒pEGFP-STAT1转染ST细胞后,West ern bl ot检测到特异性的目的蛋白;ST细胞被TGEV感染后,STAT1的m RNA表达起始于6 h,到12 h一直持续表现为低表达,于24 h时达到对照组的6.8倍,为峰值。结果提示,猪STAT1在进化上相对保守,构建重组质粒能在ST细胞中成功表达,且STAT1信号通路在TGEV感染细胞后被激活。本试验为进一步研究猪STAT1的基因在机体中的表达与调控提供了参考依据。
The purpose of this study is to study the status of STAT1 gene in St cells.In this study,STAT1 genes of several species in NCBI were used as reference,and the conserved regions of the sequences after comparison were used as primers for design and synthesis.RT-PCR was used to amplify the full fragment of pig STAT1 gene.Sequence alignment and genetic evolution analysis of target genes.The pegfp-statl recombinant plasmid was correctly sequenced by xhol and BamH I endonuclease digestion methods.The recombinant plasmid was isolated and purified.Under appropriate conditions,it was combined with pEGFP-Nl vector treated by the same method in vitro to construct the recombinant plasmid containing the target gene.Double enzyme digestion,sequencing,transfection of St cells and Western blot were used to detect the expression of target gene protein.The expression of STAT1 in St cells was detected by qPCR.The results showed that the size of pig STAT1 gene was 2274 kp;After St cells were transfected with recombinant eukaryotic plasmid pegfp-statl,the specific target protein was detected by Western blot;After infection with TGEV,the expression of STATI mRNA in St cells began at the 6th hour and continued to the 12th hour,and increased at the 24th hour,which was 6.8 times of that in the control group.The results showed that pig STAT1 was relatively conserved in evolution.The constructed recombinant plasmid could be successfully expressed in St cells and activated STATI signaling pathway after TGEV infection.This study provides a reference and basis for further study on the expression and regulation of STAT1 gene in pigs.
作者
梁青青
程慧芳
申志超
张红垒
LIANG Qing-qing;CHENG Hui-fang;SHEN Zhi-chao;ZHANG Hong-lei(Ruyang County Agricultural Technology Extension Centre,Luoyang Henan 450002,China;The College of Veterinary Medicine,Henan Agricultural University,Henan Agricultural University,Zhengzhou,Henan 450002,China;Ruyang County A gricultural Product Quality and Safety Monitoring Station,Luoyang Henan 450002,China)
出处
《当代畜牧》
2021年第9期31-35,共5页
Contemporary Animal Husbandry
作者简介
第一作者:梁青青,1992年出生,女,汉族,河南省三门峡人,助理兽医师,硕士研究生,主要研究方向为动物分子病毒学及免疫学;通讯作者:张红垒,1986年出生,男,汉族,副教授,主要研究方向为动物病毒学与免疫学。
