CAJ | 학술논문

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432 bp；SDS-PAGE分析显示，融合蛋白大小约为34．9 ku，在30℃条件下，以0．3 mmol／L的IPTG诱导6 h，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。
위획득산양IFN-γ중조단백，제취산양외주혈림파세포총 RNA，반전록득도 cDNA，이차위모판경PCR확증획득IFN-γ적ORF구，구건중조극륭재체。경PCR확증득도편마산양IFN-γ성숙태적기인서렬，련입중조표체재체 pET-32a，전입 BL21（Codon Plus）감수태세포중，측서병분석。중조표체재체경IPTG유도후，대산물진행SDS-PAGE분석，병과얼주순화。용획득적순화단백면역가토제비항체。결과현시，편마산양IFN-γ성숙태부분적편단장432 bp；SDS-PAGE분석현시，융합단백대소약위34．9 ku，재30℃조건하，이0．3 mmol／L적IPTG유도6 h，가득도대량가용성표체적목적단백；간접 ELISA측정제득적다극륭항체효개위1∶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.