犬新孢子虫吉林株GRA7基因的克隆及原核表达
摘要
新孢子虫病是目前危害养牛业健康发展的主要原虫病,该病主要造成孕牛流产、死胎以及新生犊牛的运动神经系统障碍。目前,国内外对新孢子虫病的防控尚无有效的方法。因此,筛选抗原基因,制备基因工程疫苗预防新孢子虫病的发生与发展仍是当前的首要问题。
GRA7基因是犬新孢子虫的主要致密颗粒蛋白基因,致密颗粒蛋白具有较好的免疫原性,可作为疫苗的免疫原来预防新孢子虫病。本试验根据GenBank(AF176649)上发表的犬新孢子虫GRA7基因序列设计一对含有Xho I和EcoRⅠ双酶切位点的特异性引物,PCR扩增犬新孢子虫吉林株GRA7基因,纯化PCR产物后,克隆至pMD18-T simple载体上,进行PCR鉴定、酶切鉴定及序列分析,并与GenBank中已发表的序列进行同源性比较及抗原蛋白预测分析。将鉴定正确的GRA7基因与pGEX-4T-1表达载体连接,构建GRA7基因原核表达载体,在大肠杆菌中经IPTG诱导表达,应用SDS-PAGE和Western blotting技术检测表达蛋白的反应原性。
结果表明,PCR扩增出大小为701 bp的GRA7基因片断；所克隆的犬新孢子虫吉林株GRA7基因与已发表的序列同源性达98%,经抗原蛋白预测分析该基因表达的蛋白抗原指数较高；将GRA7基因定向克隆到pGEX-4T-1表达载体中,经酶切鉴定和PCR鉴定,证明成功构建了原核表达质粒pGEX-GRA7;经SDS-PAGE和Western blotting检测证实,所表达出来的GRA7融合蛋白分子量约为51 kDa,该蛋白可被新孢子虫病阳性血清所识别,具有较好的反应原性。本试验为犬新孢子虫GRA7基因工程疫苗的研究奠定了基础。
Neosporosis is the main protozoosis against the healthy development of cattle industry.The disease mainly causes abortion in pregnant cattle, stillbirth and motor system disorders in newborn calves. Currently, there are no effective methods to prevent the disease at home and abroad. Therefore, the most important issue is the screening of antigen gene and the preparation of new genetically engineered vaccine against the occurrence and development of the disease.
GRA7 gene is a gene of Neospora caninum major dense granule protein which has good immunogenicity. The protein can be used as the vaccine to prevent Neosporosis. Based on the GRA7 gene sequence of Neospora caninum in Genbank (AF132217), a pair of specific primers with Xho I and EcoR I restriction sites were designed. Neospora caninum Jilin strain GRA7 gene was amplified by PCR, cloned into pMD18-T simple vector, identified by PCR and double-digestion, analysised by its sequence, compared with the published sequence in GenBank and analysised by the antigenic of its predicted protein. The correct GRA7 gene was connected with pGEX-4T-1 expression vector to construct GRA7 prokaryotic expression vector and induced in E.coli by IPTG The reactogenicity of the protein was detected by SDS-PAGE and Western blotting.
The results indicated that a gene fragment which is 701bp in length was amplified, which shared 98% homology with the published sequence. The prediction of the antigenic protein showed that the antigen index of the protein expressed by the gene was high. GRA7 gene was cloned into pGEX-4T-1 expression vector. Identified by PCR and double-digestion, the prokaryotic expression plasmid pGEX-GRA7 was constructed successfully. SDS-PAGE and Western blotting showed that the molecular weight of GRA7 fusion protein is about 51 kDa, the protein can be recognized by Neosporosis positive sera and the protein has a good reactogenicity. The experiment lay the foundation for the research of Neospora caninum GRA7 genetically engineered vaccine.
GRA7基因是犬新孢子虫的主要致密颗粒蛋白基因,致密颗粒蛋白具有较好的免疫原性,可作为疫苗的免疫原来预防新孢子虫病。本试验根据GenBank(AF176649)上发表的犬新孢子虫GRA7基因序列设计一对含有Xho I和EcoRⅠ双酶切位点的特异性引物,PCR扩增犬新孢子虫吉林株GRA7基因,纯化PCR产物后,克隆至pMD18-T simple载体上,进行PCR鉴定、酶切鉴定及序列分析,并与GenBank中已发表的序列进行同源性比较及抗原蛋白预测分析。将鉴定正确的GRA7基因与pGEX-4T-1表达载体连接,构建GRA7基因原核表达载体,在大肠杆菌中经IPTG诱导表达,应用SDS-PAGE和Western blotting技术检测表达蛋白的反应原性。
结果表明,PCR扩增出大小为701 bp的GRA7基因片断；所克隆的犬新孢子虫吉林株GRA7基因与已发表的序列同源性达98%,经抗原蛋白预测分析该基因表达的蛋白抗原指数较高；将GRA7基因定向克隆到pGEX-4T-1表达载体中,经酶切鉴定和PCR鉴定,证明成功构建了原核表达质粒pGEX-GRA7;经SDS-PAGE和Western blotting检测证实,所表达出来的GRA7融合蛋白分子量约为51 kDa,该蛋白可被新孢子虫病阳性血清所识别,具有较好的反应原性。本试验为犬新孢子虫GRA7基因工程疫苗的研究奠定了基础。
Neosporosis is the main protozoosis against the healthy development of cattle industry.The disease mainly causes abortion in pregnant cattle, stillbirth and motor system disorders in newborn calves. Currently, there are no effective methods to prevent the disease at home and abroad. Therefore, the most important issue is the screening of antigen gene and the preparation of new genetically engineered vaccine against the occurrence and development of the disease.
GRA7 gene is a gene of Neospora caninum major dense granule protein which has good immunogenicity. The protein can be used as the vaccine to prevent Neosporosis. Based on the GRA7 gene sequence of Neospora caninum in Genbank (AF132217), a pair of specific primers with Xho I and EcoR I restriction sites were designed. Neospora caninum Jilin strain GRA7 gene was amplified by PCR, cloned into pMD18-T simple vector, identified by PCR and double-digestion, analysised by its sequence, compared with the published sequence in GenBank and analysised by the antigenic of its predicted protein. The correct GRA7 gene was connected with pGEX-4T-1 expression vector to construct GRA7 prokaryotic expression vector and induced in E.coli by IPTG The reactogenicity of the protein was detected by SDS-PAGE and Western blotting.
The results indicated that a gene fragment which is 701bp in length was amplified, which shared 98% homology with the published sequence. The prediction of the antigenic protein showed that the antigen index of the protein expressed by the gene was high. GRA7 gene was cloned into pGEX-4T-1 expression vector. Identified by PCR and double-digestion, the prokaryotic expression plasmid pGEX-GRA7 was constructed successfully. SDS-PAGE and Western blotting showed that the molecular weight of GRA7 fusion protein is about 51 kDa, the protein can be recognized by Neosporosis positive sera and the protein has a good reactogenicity. The experiment lay the foundation for the research of Neospora caninum GRA7 genetically engineered vaccine.
引文
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