长白猪肿瘤坏死因子α成熟肽基因克隆、表达及其表达产物活性检测

Cloning,Expression of the Mature TNF-α Gene from Danish Landrance Porcine and Biological Activities Detection of the Expressed Protein

本研究旨在克隆和表达猪肿瘤坏死因子成熟肽基因及其表达蛋白的生物活性。根据GenBank登录的猪肿瘤坏死因子α(TNF-α)成熟肽基因序列设计一对上下游引物。应用RT-PCR技术直接从丹系长白猪脾脏组织中扩增猪的TNF-α成熟肽基因。将RT-PCR扩增到的特异性片段克隆到pGEM-T Easy载体中,构建pGEM-TNF重组质粒,经宝生物(大连)有限公司测序,该TNF-α成熟肽基因的长度为465 bp,编码154 aa。以重组克隆质粒pGEM-TNF为模板,PCR扩增猪TNF-α成熟肽基因,并将其插入原核表达载体pET-28a(+)中,在大肠杆菌(E.coli)BL21中诱导表达。SDS-PAGE电泳结果表明表达的融合蛋白约为38 ku,重组蛋白以包涵体形式表达,表达重组蛋白约占菌体总蛋白的36.8%。细胞毒T细胞试验表明所表达的蛋白经初步纯化、变性复性后,可明显增强淋巴毒性T细胞作用。结果提示长白猪肿瘤坏死因子得到了成功克隆与表达,表达的蛋白产物具有一定的生物活性。

The complete open reading frame（ORF） of TNF-α gene was amplified from Danish Landrace porcine spleen lymphocyte stimulated by ConA,which includes 465 bp and encodes 154 aa.The gene encoding mature TNF-α protein was cloned directionally into prokaryotic expression vector pET-28a（＋） and the fusion expression was induced in E.coli BL21.SDS-PAGE demonstrated that the fusion protein expressed in form of inclusion body is approximately 38 kDa,the product of expression account 36.8% of the total bacterium proteins.Western-blotting analysis indicated that there is a specific e1ectrophoresis strip at the place where the relative molecular mass is about 38 kDa.A 465 bp specific fragment strip was observed by detection after the recombinant bacteria was transcribed.After the protein was purified roughly and renatured,the killing effect of CTLs Cytotoxity assay confirmed that it can enhance lymphocyte cytotoxity obviously.