柔嫩艾美耳球虫(E.tenella)BJ株TA4基因的克隆和序列分析

CLONING AND SEQUENCING OF TA4 GENE OF EIMERIA TENELLA BJ STRAIN

对柔嫩艾美耳球虫(E.tenella)BJ株TA4基因进行了克隆和测序分析。以纯化的E.tenellaBJ株7h孢子化卵囊的总RNA为模板,根据国外报道的序列设计一对引物,用RT-PCR方法扩增出BJ虫株的TA4基因。用常规基因克隆方法把BJ株TA4基因插入pGEM-T克隆载体,选取正方向插入的一个阳性克隆进行酶切分析及插入片段的全序列测序分析。结果表明:该序列全长1227个核苷酸,有一个含230个密码子的开放性读框,可编码分子量约为25kDa的多肽。其后紧随533bp的3'端非编码序列。TA4-BJ与国外株TA4比较,同源性99%,突变的4个核苷酸中,2个为有义突变,使其推测氨基酸Asp变为Gly。

TA4 gene of E. tenella BJ strain infecting chickens in Beijing was cloned and sequenced. E. tenella total RNA was extracted from 7h sporulating oocysts and used as a template for cDNA synthesis after reverse transcription. TA4 cDNA was amplified by means of polymerase chain reaction(PCR). The PCR products were checked by agrose gel electrophoresis and then cloned into pGEM T easy Plasmid vector successfully. The TA4 spicific cDNA recombinant plasmids were analysed with Restriction Endonucleases (RE)(HincⅡ、HpaⅠ、NcoⅠ、ScaⅠ、SinⅠ、XmnⅠ and EcoRⅠ)digestion. Nucleotide sequence was determined by dideotide chain termination method. The results indicated that TA4 BJ gene is 1 227bp in lenghth and includes opening read frame which code for a polypeptide of 230 amino acid(molecular weight 25kDa). Comparison of TA4 BJ with the published nucleotide sequence of TA4 demonstrates they have the sequence homologies of 99%. Comparing its deduced amino acid sequence with reported amino acid sequence of TA4 demonstrated that there are 2amino acids changed with Gly from Arp and Leu from Phe.