摘要
应用异硫氰酸胍-酚-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ShTatl.3和 ShTatl.5总 RNA,在含有甲醛的琼脂凝胶电泳后,转印到硝酯纤维膜上.根据布氏锥虫 VS-Gm RNA3′端保守序列,设计合成了一个互补于它的含18个碱基的寡恢苷酸5′-GGTGTTAAAATA-TATCAG-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18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′-GAACAGTTTCTGTACTATATTG-3′.对伊氏锥虫安徽株克降虫体的二个变体 ShTatl.3和 ShTatl.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 ShTatl.3的 VSG 基因为1.7kb,而ShTatl.5的 VSG 基因则为1.4kb.
ShTatl.3 and ShTatl.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 T.brucei VSG mRNA,we designed and synthe- sized a sequence of 18 oligonucleotide 5'-GGTGTTAAAATATATCAG-3',which are com- plementary to the conserved sequences.The oligonucleotide was labeled with^(32)p.Northern blot first proved the same conserved sequences existing in T.evansi.The first strand of cDNA were synthesized successully with the 18 oligonucleotide as a primer,a 1.5 kb size dominant strip DNA all can be amplyfied from eDNA 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'-GAACAGTTTCTG- TACTATATTG-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 1.7 kb and 1.4 kb respectively.
出处
《中国农业大学学报》
CAS
CSCD
北大核心
1998年第S2期1-4,共4页
Journal of China Agricultural University
关键词
伊氏锥虫
VSG基因
分离
Trypanosoma evansi
VSG gene
isolation
