RT-PCR法分离伊氏锥虫VSG基因的研究  

ISOLATION OF VSG GENE OF TRYPANOSOMA EVANSI BY RT-PCR

作　　者：周金林[1] 沈杰[1] 

机构地区：[1]中国农科院上海家畜寄生虫病研究所,上海200232

出　　处：《畜牧兽医学报》2000年第3期277-280,共4页ACTA VETERINARIA ET ZOOTECHNICA SINICA

摘　　要：应用异硫氰酸胍一酚一氯仿一步法分离伊氏锥虫安徽株克隆虫体ShTatl的二个抗原变异体ShTat1 3和ShTat1 5总RNA ,在含有甲醛的琼脂糖凝胶电泳后 ,转印到硝酸纤维膜上。根据布氏锥虫VSGmRNA3’端保守序列 ,设计合成了一个互补于它的含十八个碱基的寡核苷酸 5’—GGTGTTAAAATATATCAG— 3’ ,经32 P标记 ,Northern杂交 ,首次证明伊氏锥虫含有同样的保守序列。利用合成的十八碱基寡核苷酸作为反转录特异引物 ,成功合成cDNA第一链 ,以cDNA第一链作为PCR扩增模板 ,均能特异性的扩增出一条主带为 1 5kb的DNA ,与VSG基因大小相符。根据所有锥虫mRNA在 5’端的保守序列 ,设计合成了RT -PCR下游引物 5’—GAACAGTTTCTG TACTATATTG— 3’。对伊氏锥虫安徽株克隆虫体的二个变体ShTat1 3和ShTat1 5的RNA ,进行RT—PCR扩增 ,均特异性地分离出VSG基因 ,二个变异体VSG基因大小差异显著 ,其中ShTat1 3的VSG基因为 1 7kb ,而ShTat1 5的VSG基因则为 1 4kb。ShTat1 3 and ShTat1 5 are two variable antigen types of a cloning Anhui strain of Trypanosoma evansi.Total RNA of the two variable antigen types were extracted by acid guanidinium thiocyanate phenol chlorform single step method.RNA were subjected to agarose gel electrophoresis,the gels were then blotted onto nitro cellulose filters.Referred to conserved sequences of the 3’ end of Trypanosoma brucei VSG mRNA,we designed and synthesized a sequence of 18 oligonucleotide 5’ GGTGTTAAAATATATCAG 3’,which are complementary to the conserved sequences.The oligonucleotide was labeled with 32 p,Northern blot first proved the same conserved sequences existing in Trypanosoma evansi.The first strand of cDNA were synthesized successfully with the 18 oligonucleotide as a primer,a 1 5kb size dominant strip DNA all can be amplified from cDNA pool,which match with the size of VSG gene.According to 5’end constant nucleotide sequences of all trypanosome mRNA,we designed and synthesized RT PCR downstream primer 5’ GAACAGTTTCTGTACTATATTG 3’.We isolated specially VSG gene of ShTat1 3 and ShTat1 5 by RT PCR.VSG gene of the two variable antigen types differ obviously in size,the size of ShTat1 3 and ShTat1 5 VSG gene is 1 7kb and 1 4kb respectively.

关 键 词：伊氏锥虫 VSG基因 分离 RT-PCR 抗原变异 

分 类 号：S855.912.9[农业科学—临床兽医学]