中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2008年
9期
1184-1186
,共3页
刘俊杰%郑骏年%吴洋%李望%郑宏祥%温儒民%刘晓筠%毛立军%裴冬生%孔德领
劉俊傑%鄭駿年%吳洋%李望%鄭宏祥%溫儒民%劉曉筠%毛立軍%裴鼕生%孔德領
류준걸%정준년%오양%리망%정굉상%온유민%류효균%모립군%배동생%공덕령
肾细胞癌%抗原%蛋白多糖
腎細胞癌%抗原%蛋白多糖
신세포암%항원%단백다당
Renal cell carcinoma%Antigen%Protcoglycan
目的 探讨肾癌肿瘤相关抗原G250及G250蛋白多糖(PG)区基因的克隆,蛋白表达及鉴定.方法 从肾癌细胞786-0中提取mRNA,采用逆转录-聚合酶链反应(RT-PCR)扩增出G250及G250 PC,区cDNA.将获得的cDNA片段插入pET28a(+)表达载体,构建重组质粒pET-28a(+).G250/pET-28a(+)-G250 PG,转化DH5α大肠杆菌.通过PCR鉴定筛选出正确的重组子,并用其转化BL21 Codon Plus菌.采用IPTG诱导工程菌进行诱导表达,SDS-PAGE及Westernblot鉴定.结果 克隆的基因经测序证实,G250/G250 PG区序列正确.构建重组质粒pET-28a(+).G250/pET-28a(+)-G250 PG,并在原核系统中表达G250/G250 PC重组蛋白,目的 蛋白均具有较好的抗原性和特异性.结论 成功完成G250/G250 Pc区基因克隆及表达,为在G250/G250PG蛋白纯化基础上进行抗体制备和G250功能研究提供了实验依据.
目的 探討腎癌腫瘤相關抗原G250及G250蛋白多糖(PG)區基因的剋隆,蛋白錶達及鑒定.方法 從腎癌細胞786-0中提取mRNA,採用逆轉錄-聚閤酶鏈反應(RT-PCR)擴增齣G250及G250 PC,區cDNA.將穫得的cDNA片段插入pET28a(+)錶達載體,構建重組質粒pET-28a(+).G250/pET-28a(+)-G250 PG,轉化DH5α大腸桿菌.通過PCR鑒定篩選齣正確的重組子,併用其轉化BL21 Codon Plus菌.採用IPTG誘導工程菌進行誘導錶達,SDS-PAGE及Westernblot鑒定.結果 剋隆的基因經測序證實,G250/G250 PG區序列正確.構建重組質粒pET-28a(+).G250/pET-28a(+)-G250 PG,併在原覈繫統中錶達G250/G250 PC重組蛋白,目的 蛋白均具有較好的抗原性和特異性.結論 成功完成G250/G250 Pc區基因剋隆及錶達,為在G250/G250PG蛋白純化基礎上進行抗體製備和G250功能研究提供瞭實驗依據.
목적 탐토신암종류상관항원G250급G250단백다당(PG)구기인적극륭,단백표체급감정.방법 종신암세포786-0중제취mRNA,채용역전록-취합매련반응(RT-PCR)확증출G250급G250 PC,구cDNA.장획득적cDNA편단삽입pET28a(+)표체재체,구건중조질립pET-28a(+).G250/pET-28a(+)-G250 PG,전화DH5α대장간균.통과PCR감정사선출정학적중조자,병용기전화BL21 Codon Plus균.채용IPTG유도공정균진행유도표체,SDS-PAGE급Westernblot감정.결과 극륭적기인경측서증실,G250/G250 PG구서렬정학.구건중조질립pET-28a(+).G250/pET-28a(+)-G250 PG,병재원핵계통중표체G250/G250 PC중조단백,목적 단백균구유교호적항원성화특이성.결론 성공완성G250/G250 Pc구기인극륭급표체,위재G250/G250PG단백순화기출상진행항체제비화G250공능연구제공료실험의거.
Objective To clone,express and identify the tumor-associated antigen G250 and G250 pmteoglyean(PC)region.Methods The mRNA Was extracted from human renal carcinoma cellline 786-0.Gene fragments eneoding G250 and G250 PG were obtained by RT-PCR.and cloned into prokaryotie expression vector pET28a(+)to construct the recombinant plasmid pET-28a(+)-G250/pET-28a(+)-G250 PG.The recombinant vectors were transfected into E.coli DH5α and PCR assay was performed to find out the right recombinant clones.The right clones were transfected into the expression host (E.coli BL21 Codon Plus).and the engineering bacteria were indueed by IPTG.The results were acquired by SDS-PAGE and Western blot.Resnits DNA sequence analysis showed that the sepuence edconding G250/G250 PG region was the same as that in GenBank.Gene of G250 and G250 PG region Was expressed in E.coli BL21 Codon Plus successfully.Western blot analysis showed that the two kinds of recombinant protein could be specially recognized by their corresponding antibodies.They both had better antigenicity and specificity.Conclusion This study provides experimental basis for the purification of G250/G250 PG protein and the further study on G250 function and preparation of antibody.
