牛干扰素-tau基因的原核表达及生物功能鉴定

本试验采用PCR方法直接从牛早期胚胎细胞液中扩增牛干扰素-tau （bIFN-τ）基因（含信号肽基因序列），并克隆到pGEM-T载体中，筛选阳性克隆，将含信号肽和不含信号肽的bIFN-τ 片断分别克隆到原核表达载体pET-30a(+)，构建重组质粒，进行IPTG诱导表达，表达产物进行变性、复性和纯化，最后通过抗病毒活性检测和对体外培养的牛子宫内膜上皮细胞形态变化的影响，鉴定其生物学功能。结果表明，不提取胚胎基因组DNA，以胚胎的裂解液为模板，可以从少量（5枚）牛囊胚中直接扩增得到bIFN-τ基因，与Genbank报道的序列相比，核苷酸和氨基酸的同源性分别达99％和97％；SDS-PAGE电泳检测，不含信号肽的bIFN-τ基因与表达载体形成的重组质粒，在约20kD处出现特异性条带，与目标蛋白分子量一致；病毒抑制法测定rbIFN-τ的抗病毒活性单位为1×104IU/mg，在牛子宫内膜上皮细胞培养液中添加rbIFN-τ作用24 h后，细胞体积增大，细胞内出现明显的囊泡状结构，表明本研究获得的rbIFN-τ具有一定的生物活性。

Expression of Bovine Interferon-tau in Escherichia coli and Identification of Its Biological Activities

In this study, the gene encoding for bovine interferon-tau (bIFN-τ), with signal sequence, was obtained through PCR from bovine early embryos and subcloned into pGEM-T vector. After being verified, the bIFN-τ fragments with signal sequence or without signal sequence were inserted into the expression vector pET-30a(+), respectively. Two recombinant plasmids were induced to express the recombinant proteins by Isopropyl β-D-1-thiogalactopyranoside, respectively. The expressed products were then denatured, renatured and purified. The biological activities of purified rbIFN-τ were identified by assaying its antiviral activity and its effect on the morphology of bovine endometrial epithelial cells during in vitro culture. The results showed that bIFN-τ gene could be obtained from five bovine blastocysts by PCR without extraction of genomic DNA. Its homologies were 99% in nucleotide acids and 97% in amino acids to the sequence in GenBank (XM593584). The products of rbIFN-τ with minus signal sequence expressed in pET-30a(+) were analyzed by SDS-PAGE, and a new protein of 20kD was detected. Its molecular weight was as the same as the designed. The antiviral activity of rbIFN-τ was 1×104IU/mg using standard cytopathic reduction assay. The rbIFN-τ induced obvious morphological changes in bovine endometrial epithelial cells in vitro. The cell volume was larger than the control and a lot of vesicles appeared in the cytoplasms after 24 h culture in presence of 2.9µg/ml rbIFN-τ. In conclusion, the purified rbIFN-τ with biological activities was obtained in this experiment.