瑟氏泰勒虫p33基因在酵母菌中的表达
发布时间：2018-10-12 12:21
【摘要】：瑟氏泰勒虫(Theileriasergenti)是寄生于牛体内的一种血液原虫,是泰勒虫科、泰勒虫属,主要寄生于牛的淋巴细胞、红细胞、巨噬细胞中,通过蜱进行传播。现阶段,随着养牛业的不断发展,牛的瑟氏泰勒虫病近几年发生频率呈现上升趋势,对养牛业的危害极大。但到目前为止还没有特效药物用于瑟氏泰勒虫病的治疗和预防,而且尚无该病的疫苗。瑟氏泰勒虫p33基因,是该虫体的主要表面蛋白基因,具有较好的免疫原性和反应原性,认为是较理想的候选抗原。真核表达系统,除了具备原核表达系统的优点外,还具有表达外源蛋白量高、遗传性状稳定、产物提纯简单等诸多优点,受到越来越多的人青睐。本试验根据Genbank中T.sergentip33基因中国株序列,设计一对特异性引物,用常规PCR扩增出大小为786 bp的p33目的基因片段,将其克隆到pMD-19-T载体上,构建pMD-19-T-p33重组克隆质粒,经PCR、双酶切鉴定测序分析,证实目的片段正确地克隆到pMD-19-T载体上。然后将p33基因亚克隆到酵母表达载体pPICZaA上,重组质粒经PCR、EcoRⅠ和XbaⅠ双酶切鉴定,证实成功构建含有目的基因片段的真核表达载体pPICZα A-p33。然后将其转化到GS115酵母感受态细胞中,最终通过800μg/ml浓度的博来霉素筛选得到高拷贝重组子,经PCR筛选阳性菌株后,通过最终浓度为1%的甲醇诱导并在毕赤酵母中大量表达外源蛋白,然后对诱导产物进行SDS-PAGE,得到大小为29.67Ku的蛋白,与预期结果相符合。而后对诱导蛋白进行Western blot反应原性鉴定,蛋白可以被牛瑟氏泰勒虫的阳性血清特异性识别,表明得到的蛋白具有较好的反应原性。本试验结果为以后瑟氏泰勒虫病诊断试剂盒、瑟氏泰勒虫p33基因的单克隆抗体和瑟氏泰勒虫病疫苗研究等研究打下坚实的基础。
[Abstract]:(Theileriasergenti) is a blood protozoa parasitic in cattle. It is mainly parasitic on bovine lymphocytes, erythrocytes and macrophages, and is transmitted by ticks. At present, with the continuous development of cattle industry, the frequency of Taylor's disease in cattle is increasing in recent years, which is harmful to cattle industry. But so far there is no specific drug for the treatment and prevention of Taylor's disease, and there is no vaccine for the disease. The p33 gene is the main surface protein gene of the worm, and has good immunogenicity and reactivity, so it is considered to be an ideal candidate antigen. Eukaryotic expression system not only has the advantages of prokaryotic expression system but also has many advantages such as high expression amount of exogenous protein stable hereditary character simple product purification and so on. According to the Chinese strain sequence of T.sergentip33 gene in Genbank, a pair of specific primers were designed. The p33 gene fragment of 786 bp was amplified by conventional PCR and cloned into pMD-19-T vector to construct the recombinant pMD-19-T-p33 clone plasmid. The target fragment was correctly cloned into pMD-19-T vector by PCR, double digestion and sequencing analysis. Then the p33 gene was subcloned into yeast expression vector pPICZaA, and the recombinant plasmid was identified by PCR,EcoR 鈪，
本文编号：2266092
[Abstract]:(Theileriasergenti) is a blood protozoa parasitic in cattle. It is mainly parasitic on bovine lymphocytes, erythrocytes and macrophages, and is transmitted by ticks. At present, with the continuous development of cattle industry, the frequency of Taylor's disease in cattle is increasing in recent years, which is harmful to cattle industry. But so far there is no specific drug for the treatment and prevention of Taylor's disease, and there is no vaccine for the disease. The p33 gene is the main surface protein gene of the worm, and has good immunogenicity and reactivity, so it is considered to be an ideal candidate antigen. Eukaryotic expression system not only has the advantages of prokaryotic expression system but also has many advantages such as high expression amount of exogenous protein stable hereditary character simple product purification and so on. According to the Chinese strain sequence of T.sergentip33 gene in Genbank, a pair of specific primers were designed. The p33 gene fragment of 786 bp was amplified by conventional PCR and cloned into pMD-19-T vector to construct the recombinant pMD-19-T-p33 clone plasmid. The target fragment was correctly cloned into pMD-19-T vector by PCR, double digestion and sequencing analysis. Then the p33 gene was subcloned into yeast expression vector pPICZaA, and the recombinant plasmid was identified by PCR,EcoR 鈪，
本文编号：2266092
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