中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY
2013年
4期
301-303
,共3页
郭静霞%魏红山%宋永继%赵静%刘爱霞%刘佳%陈霖%徐军%李伯安
郭靜霞%魏紅山%宋永繼%趙靜%劉愛霞%劉佳%陳霖%徐軍%李伯安
곽정하%위홍산%송영계%조정%류애하%류가%진림%서군%리백안
高尔基体%蛋白质73%克隆,分子%抗体,单克隆%肝肿瘤
高爾基體%蛋白質73%剋隆,分子%抗體,單剋隆%肝腫瘤
고이기체%단백질73%극륭,분자%항체,단극륭%간종류
Golgi apparatus%Protein P73%Clone molecular%Antibodies monoclonal%Liver neoplasms
目的 克隆、表达高尔基体蛋白73(GP73),并制备单克隆抗体.方法 用逆转录聚合酶链反应法从人肝癌细胞系HepG2细胞中扩增GP73编码序列,将产物与pQE31载体连接,转化人大肠埃希菌BL21,诱导表达后通过His标签进行纯化,用纯化后的蛋白免疫BALB/c小鼠,采用常规的细胞融合技术制备单克隆抗体,并对其进行鉴定.结果 重组载体经序列分析与报道序列同源性100％,并获得了重组蛋白.筛选出5株能稳定分泌抗GP73的单克隆抗体杂交瘤细胞株,免疫球蛋白类型有2株为IgG1类.Western Blot印迹显示这些单抗能特异结合GP73蛋白.结论 成功构建了人高尔基体蛋白73原核表达体系,并获得了单克隆抗体.
目的 剋隆、錶達高爾基體蛋白73(GP73),併製備單剋隆抗體.方法 用逆轉錄聚閤酶鏈反應法從人肝癌細胞繫HepG2細胞中擴增GP73編碼序列,將產物與pQE31載體連接,轉化人大腸埃希菌BL21,誘導錶達後通過His標籤進行純化,用純化後的蛋白免疫BALB/c小鼠,採用常規的細胞融閤技術製備單剋隆抗體,併對其進行鑒定.結果 重組載體經序列分析與報道序列同源性100％,併穫得瞭重組蛋白.篩選齣5株能穩定分泌抗GP73的單剋隆抗體雜交瘤細胞株,免疫毬蛋白類型有2株為IgG1類.Western Blot印跡顯示這些單抗能特異結閤GP73蛋白.結論 成功構建瞭人高爾基體蛋白73原覈錶達體繫,併穫得瞭單剋隆抗體.
목적 극륭、표체고이기체단백73(GP73),병제비단극륭항체.방법 용역전록취합매련반응법종인간암세포계HepG2세포중확증GP73편마서렬,장산물여pQE31재체련접,전화인대장애희균BL21,유도표체후통과His표첨진행순화,용순화후적단백면역BALB/c소서,채용상규적세포융합기술제비단극륭항체,병대기진행감정.결과 중조재체경서렬분석여보도서렬동원성100％,병획득료중조단백.사선출5주능은정분비항GP73적단극륭항체잡교류세포주,면역구단백류형유2주위IgG1류.Western Blot인적현시저사단항능특이결합GP73단백.결론 성공구건료인고이기체단백73원핵표체체계,병획득료단극륭항체.
Objective To clone and express human Golgi glycoprotein73 protein,and prepare the monoclonal antibody (mAb) against the protein.Methods GP73 gene was amplified from HepG2 cells by RT-PCR,then ligated with pQE31 to form recombinant plasmid pQE-GP73 and transformed into E.coli BL21.The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice.The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique.Western Blot was used to detect specificity of mAbs.Results The prokaryotic plasmid expressing the recombinant protein was constructed,and the GP73 recombinant protein was expressed and purified.Five hybridoma cell lines that secreted anti-GP73 mAbs were obtained.2 of 5 mAbs were the IgG1 subtype.Western Blot indicated the mAbs showed specific combination with GP73 protein.Conclusion The GP73 recombinant protein is highly purified and has strong antigenicity.The anti-GP73 mAbs were prepared successfully.
