山羊IFN-γ基因的克隆表达与多克隆抗体制备  

Cloning and Expression of Goat IFN-γGene and Preparation of Polyclonal Antibodies

作　　者：安贝[1] 赵玄多 杨雯昱[1] 侯伟杰[1] 高洋[1] 陈德坤[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《动物医学进展》2014年第11期29-34,共6页Progress In Veterinary Medicine

基　　金：陕西省科技计划项目(2013KTZB02-02-02)

摘　　要：为获得山羊IFN-γ重组蛋白,提取山羊外周血淋巴细胞总RNA,反转录得到cDNA,以此为模板经PCR扩增获得IFN-γ的ORF区,构建重组克隆载体。经PCR扩增得到编码山羊IFN-γ成熟肽的基因序列,连入重组表达载体pET-32a,转入BL21(Codon Plus)感受态细胞中,测序并分析。重组表达载体经IPTG诱导后,对产物进行SDS-PAGE分析,并过镍柱纯化。用获得的纯化蛋白免疫家兔制备抗体。结果显示,编码山羊IFN-γ成熟肽部分的片段长432bp;SDS-PAGE分析显示,融合蛋白大小约为34.9ku,在30℃条件下,以0.3mmol/L的IPTG诱导6h,可得到大量可溶性表达的目的蛋白;间接ELISA测定制得的多克隆抗体效价为1∶106。To obtain the expression products of goat IFN-γ,total RNA was extracted from activated goat PBMCs,and cDNA was synthesized and used as template for PCR.The recombinant cloning vector was constructed.The sequence encoding mature peptide region was cloned by PCR and inserted into the ex-pression vector pET-32a,and the recombinant plasmid was transformed into the competent cell BL21 （Co-don Plus）,followed by sequencing and analysis.After induced by IPTG,the expression products were and analysized by SDS-PAGE and purified by Ni-NTA.The purified protein was immunize rabbits to prepare polyclonal antibodies.Sequence analysis suggested that sequence encoding the mature peptide was 432 bp. SDS-PAGE indicated that the fusion protein was about 34.9 ku,and a large amount of soluble protein could be expressed when induced with a total concentration of 0.3 mmol/L/L IPTG for 6 h at 30℃.The titter of polyclonal antibodies was 1∶106 detected by indirect ELISA.

关 键 词：山羊 IFN-Γ 基因克隆 原核表达 抗体制备 

分 类 号：S852.4[农业科学—基础兽医学]