中华医学杂志(英文版)
中華醫學雜誌(英文版)
중화의학잡지(영문판)
CHINESE MEDICAL JOURNAL
2002年
7期
1064-1069
,共6页
逆转录病毒%单链抗体%肿瘤
逆轉錄病毒%單鏈抗體%腫瘤
역전록병독%단련항체%종류
retroviral vector%single-chain variable antibody fragment%cancer
目的将单链抗体基因插入单嗜性逆转录病毒胞膜蛋白，以构建能靶向感染肿瘤细胞的逆转录病毒载体。方法 构建单链抗体融合表达质粒PET20b-scFv，通过酶联免疫分析，Western blot检测基因工程单链抗体的抗原结合活性。利用PCR定点突变技术，在野生型MoMulv胞膜蛋白上的SU亚单位氨基末端产生合适的酶切位点，插入体外构建，证实具有抗原亲合活性的抗人脑胶质瘤细胞膜抗原的单链抗体基因，并构建利用CMV启动子高效表达胞膜蛋白-单链抗体融合基因的表达质粒，转染Ψ2包装细胞后，以lac-z为报告基因，包装出重组逆转录病毒，进行病毒结合实验，感染实验。结果 酶联免疫分析，Western blot 结果证实基因工程单链抗体能与肿瘤细胞表面相应抗原结合，病毒结合实验，感染实验，结果证明所构建嵌合型逆转录病毒能改变其嗜性，由仅感染鼠细胞到特异性感染人脑胶质瘤细胞，且这种作用能为特异性单克隆抗体阻断。结论 利用单链抗体修饰单嗜性逆转录病毒胞膜蛋白的SU亚单位能达到选择性感染肿瘤细胞的目的。
目的將單鏈抗體基因插入單嗜性逆轉錄病毒胞膜蛋白，以構建能靶嚮感染腫瘤細胞的逆轉錄病毒載體。方法 構建單鏈抗體融閤錶達質粒PET20b-scFv，通過酶聯免疫分析，Western blot檢測基因工程單鏈抗體的抗原結閤活性。利用PCR定點突變技術，在野生型MoMulv胞膜蛋白上的SU亞單位氨基末耑產生閤適的酶切位點，插入體外構建，證實具有抗原親閤活性的抗人腦膠質瘤細胞膜抗原的單鏈抗體基因，併構建利用CMV啟動子高效錶達胞膜蛋白-單鏈抗體融閤基因的錶達質粒，轉染Ψ2包裝細胞後，以lac-z為報告基因，包裝齣重組逆轉錄病毒，進行病毒結閤實驗，感染實驗。結果 酶聯免疫分析，Western blot 結果證實基因工程單鏈抗體能與腫瘤細胞錶麵相應抗原結閤，病毒結閤實驗，感染實驗，結果證明所構建嵌閤型逆轉錄病毒能改變其嗜性，由僅感染鼠細胞到特異性感染人腦膠質瘤細胞，且這種作用能為特異性單剋隆抗體阻斷。結論 利用單鏈抗體脩飾單嗜性逆轉錄病毒胞膜蛋白的SU亞單位能達到選擇性感染腫瘤細胞的目的。
목적장단련항체기인삽입단기성역전록병독포막단백，이구건능파향감염종류세포적역전록병독재체。방법 구건단련항체융합표체질립PET20b-scFv，통과매련면역분석，Western blot검측기인공정단련항체적항원결합활성。이용PCR정점돌변기술，재야생형MoMulv포막단백상적SU아단위안기말단산생합괄적매절위점，삽입체외구건，증실구유항원친합활성적항인뇌효질류세포막항원적단련항체기인，병구건이용CMV계동자고효표체포막단백-단련항체융합기인적표체질립，전염Ψ2포장세포후，이lac-z위보고기인，포장출중조역전록병독，진행병독결합실험，감염실험。결과 매련면역분석，Western blot 결과증실기인공정단련항체능여종류세포표면상응항원결합，병독결합실험，감염실험，결과증명소구건감합형역전록병독능개변기기성，유부감염서세포도특이성감염인뇌효질류세포，차저충작용능위특이성단극륭항체조단。결론 이용단련항체수식단기성역전록병독포막단백적SU아단위능체도선택성감염종류세포적목적。
Objective To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope.Methods Single-chain antibody expression vector pET -20bScfv was constructed. Binding activity of the genetically engineered single-chain variable antibody fragment was verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)-directed mutagenesis, the appropriate cloning site EcoT22 Ⅰ/Sal Ⅰ was generated at the N-terminus of receptor-binding SU domain in the MoMLV env polypetide. Then the single-chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv-env fusion gene fragment was subcloned into CMV expression vector. The Lac-Z retrovirus that co-displayed the Scfv-env chimeric protein and wild-type env protein was produced by transfection of Ψ2 cells with retroviral plasmid and the fusion gene expressing plasmid. To confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed.Results The results of ELISA and Western blot showed that the genetically engineered single-chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus-binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen.Conclusion These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.
