广州管圆线虫γ-丁基甜菜碱羟化酶基因的获得与分析

Isolation and analysis of the gene encoding for γ-butyrobetaine hydroxylase of Angiostrongylus cantonensis

目的 对广州管圆线虫成虫cDNA文库的插入片段进行研究。方法 铺平板培养广州管圆线虫成虫cDNA文库 ,从中挑选生长、分离良好的噬菌斑进行PCR扩增 ,根据PCR产物电泳结果挑选较大的cDNA插入片段进行克隆、测序和原核诱导表达 ,对表达产物进行免疫学鉴定 ,用生物信息学方法分析其结构和功能。结果 获得了国内外尚未见报道的广州管圆线虫全长基因 ,该基因长 14 16bp ,编码由 4 2 1个氨基酸组成的γ -丁基甜菜碱羟化酶 (gamma -butyrobetaine ,2 -oxoglu taratedioxygenase ,GAMMA -BBH ,EC 1 14 11 1) ;免疫学鉴定结果显示GAMMA -BBH不能与大鼠的抗血清结合。 结论 首次获得广州管圆线虫成虫GAMMA -BBH表达基因 ,为广州管圆线虫的研究提供实验室依据。

To study the inserted cDNA fragment of cDNA library of adult Angiostrongylus cantonensis,the cDNA library was cultured in BL/MgSO 4 top agar,and several recombinant bacteriophages were isolated to amplify the inserted cDNA fragments by PCR.The largest inserted one was cloned,sequenced,and analyzed by the method of bioinformatics,and the expressed products were identified by immunological methods.By using these methods,an unreported complete gene was obtained,which was 1416 bp in length and could encode the γ butyrobetaine hydroxylase(GAMMA-BBH,EC 1.14.11.1)that was composed of 421 amino acids,GAMMA-BBH could not bing with the antisera of rats.It concludes that the gene encoding for the GAMMA-BBH of Angiostrongylus cantonensis was successfully isolated for the first time,and this can provide a foundation for the further study of this parasite.