中国人兽共患病学报
中國人獸共患病學報
중국인수공환병학보
CHINESE JOURNAL OF ZOONOSES
2009年
12期
1195-1198
,共4页
王乐旬%余新炳%黄灿%刘玲%胡旭初%徐劲
王樂旬%餘新炳%黃燦%劉玲%鬍旭初%徐勁
왕악순%여신병%황찬%류령%호욱초%서경
华支睾吸虫%Ras原癌基因%生物信息学%克隆%表达
華支睪吸蟲%Ras原癌基因%生物信息學%剋隆%錶達
화지고흡충%Ras원암기인%생물신식학%극륭%표체
Clonorchis sinensis%Ras oncogene%bioinformatics%molecular cloning%expression%purification
目的 从华支睾吸虫cDNA文库中筛选Rap2B样基因并分析其结构和功能,初步进行克隆和表达纯化,为进一步研究其功能奠定基础.方法 利用多种生物信息学分析软件,分析华支睾吸虫Rap2B蛋白的拓扑学结构、生物学和免疫学功能特征.设计引物,从华支睾cDNA质粒文库中扩增目的 基因cDNA序列,构建原核重组质粒并初步表达纯化.结果 华支睾吸虫Rap2B样基因长度为847bp ,其全长编码序列为426bp , 编码141个氨基酸,理论分子量是15851.1.重组的原核表达质粒pET-28a(+)-Rap2B经PCR、双酶切及DNA测序结果均表明重组质粒构建成功.该基因在大肠杆菌中能高效表达.经His亲和层析柱获得了纯化的重组蛋白,Western blotting证实Rap2B重组蛋白能被感染华支睾吸虫的大鼠血清识别.结论 华支睾吸虫Rap2B蛋白经生物信息学预测为ras原癌基因家族成员,可能在华支睾吸虫致癌过程中有一定作用.该基因可在原核表达系统中高效表达,并得到了纯化的重组蛋白,为进一步研究该蛋白的功能以及在致病尤其是可能促发肿瘤的作用方面奠定一定基础.
目的 從華支睪吸蟲cDNA文庫中篩選Rap2B樣基因併分析其結構和功能,初步進行剋隆和錶達純化,為進一步研究其功能奠定基礎.方法 利用多種生物信息學分析軟件,分析華支睪吸蟲Rap2B蛋白的拓撲學結構、生物學和免疫學功能特徵.設計引物,從華支睪cDNA質粒文庫中擴增目的 基因cDNA序列,構建原覈重組質粒併初步錶達純化.結果 華支睪吸蟲Rap2B樣基因長度為847bp ,其全長編碼序列為426bp , 編碼141箇氨基痠,理論分子量是15851.1.重組的原覈錶達質粒pET-28a(+)-Rap2B經PCR、雙酶切及DNA測序結果均錶明重組質粒構建成功.該基因在大腸桿菌中能高效錶達.經His親和層析柱穫得瞭純化的重組蛋白,Western blotting證實Rap2B重組蛋白能被感染華支睪吸蟲的大鼠血清識彆.結論 華支睪吸蟲Rap2B蛋白經生物信息學預測為ras原癌基因傢族成員,可能在華支睪吸蟲緻癌過程中有一定作用.該基因可在原覈錶達繫統中高效錶達,併得到瞭純化的重組蛋白,為進一步研究該蛋白的功能以及在緻病尤其是可能促髮腫瘤的作用方麵奠定一定基礎.
목적 종화지고흡충cDNA문고중사선Rap2B양기인병분석기결구화공능,초보진행극륭화표체순화,위진일보연구기공능전정기출.방법 이용다충생물신식학분석연건,분석화지고흡충Rap2B단백적탁복학결구、생물학화면역학공능특정.설계인물,종화지고cDNA질립문고중확증목적 기인cDNA서렬,구건원핵중조질립병초보표체순화.결과 화지고흡충Rap2B양기인장도위847bp ,기전장편마서렬위426bp , 편마141개안기산,이론분자량시15851.1.중조적원핵표체질립pET-28a(+)-Rap2B경PCR、쌍매절급DNA측서결과균표명중조질립구건성공.해기인재대장간균중능고효표체.경His친화층석주획득료순화적중조단백,Western blotting증실Rap2B중조단백능피감염화지고흡충적대서혈청식별.결론 화지고흡충Rap2B단백경생물신식학예측위ras원암기인가족성원,가능재화지고흡충치암과정중유일정작용.해기인가재원핵표체계통중고효표체,병득도료순화적중조단백,위진일보연구해단백적공능이급재치병우기시가능촉발종류적작용방면전정일정기출.
To analyze the structural characteristics of the Rap2B gene from Clonorchis sinensis by bioinformatics and to clone and express this gene through genetic engineering methods in order to obtain the corresponding recombinant protein.The structural and functional characteristics of the Rap2B protein were analyzed by using the related bioinformatic softwares. Using the full-length cDNA plasmid clone (Noc009e03) as template , the coding region of Rap2B gene sequence was amplified with PCR and cloned into the prokaryotic expression vector pET-28a(+) and then expressed in E.coli BL21 through IPIG induction. The recombinant protein expressed was purified by Ni-IDA affinity chromatography and detected by SDS-PAGE and Western blotting. It was demonstrated that the size of this gene was 847 bp in length, encoding 141 amino acids and its theoretical molecular weight was 15851.1 daltons. As demonstrated by PCR, double enzyme digestion and DNA sequencing, the recombinant expression plasmid pET-28a(+)-Rap2B was successively constructed and the Rap2B-like gene was highly expressed in E.coli BL21. The recombinant protein was obtained through purification with His affinity chromatography and could react specifically with the serum of rats infected with C.sinensis. Through these investigations, the Rap2B protein may be predicted as a member of the Ras oncogene family protein and is potential in the carcinogenic process of C.sinensis.
