骨巨细胞瘤GCF-5抗原的cDNA克隆化及其在大肠杆菌中的表达

GCF-5 antigen's cDNA Cloning of Giant Cell Tumor of Bone and Its Expression in E. Coli.

目的 ＧＣＦ－５是骨巨细胞瘤（ＧＣＴ）特异性单抗，其抗原是了解ＧＣＴ特性的途径之一。为了利用这一途径，对ＧＣＦ－５抗原的ｃＤＮＡ进行了克隆及表达。方法 盐酸胍和有机溶剂法提取总ＲＮＡ，Ｏｌｉｇｏ（ｄＴ）－Ｃｅｌｌｕｌｏｓｅ亲和层析法分离ｍＲＮＡ，反转录合成ｃＤＮＡ，用 ｇｔ１１Ｓｆｉ－Ｎｏｔ表达载体构建ｃＤＮＡ文库，免疫学筛选并克隆ＧＣＦ－５抗原的ｃＤＮＡ，大肠杆菌Ｙ１０８９表达含ＧＣＦ－５抗原的融合蛋白，细胞免疫抑制实验证实该融合蛋白含有ＧＣＦ－５抗原成分。结果 经Ｏｌｉｇｏ（ｄＴ）－Ｃｅｌｌｕｌｏｓｅ亲和层析法分离的ｍＲＮＡ，浓度达２０．５６μｇ／ｍｌ，ＯＤ２６０／２８０比值为１．８６９，经反转录所得的ｃＤＮＡ产物从数百到数千ｂｐ之间，产物完整；构建的 ｇｔ１１Ｓｆｉ－Ｎｏｔ载体经体外包装后均呈无色透明噬斑，用抗体筛选出了一阳性噬斑，重复克隆化所得噬斑均可被抗体染色；该噬菌体感染大肠杆菌Ｙ１０８９，分别于３２℃与４２℃培养，选出溶源菌，ＩＰＴＧ诱导融合蛋白，ＳＤＳ－ＰＡＧＥ显示稍大于正常β－半乳糖苷酶的融合蛋白带，与融合蛋白预孵育后的抗体所做细胞免疫化学染色不显色。结论 构建了完整的ＧＣＴｃＤＮＡ文库；?

Objective To clone the cDNA of GCF-5 antigen and to study its expression in E. Coli.Methods Cultured GCT cells from surgical specimens were used for total RNA isolation with Guanidine HCLand organic solvents. Oligo(dT) -cellulose was used in selection of poly (A) + RNA. Then, synthesis of cDNA,ligation with synthetic EcoR I adapter, and constructing cDNA libraries with bacteriophage A gt 11 Sfi-Notvectors are carried out with Ribo Clone system. After packaging in vitro, the phages were transferred to adsorbE. Coli. Y1090. Immunoscreening of lambda expression libraries with GCF-5 McAb and further cloning wasrepeated with the same procedure. The phages from positive clone was then adsorhed to E. Coli. Y1089.Transcription initiation was regulated with IPTG at 42℃ Cyto-immunology block assay was performed toidentified the fusion proteins, which contain GCF-5 antigen. Results The concentration of poly(A)+RNApurified by Oligo (dT) -cellulose affinity reached 20. 56 g/ml. The ratio of OD260/280 is 1. 869. Gel analysisof cDNA showed it distributed from hundreds to thousands of bp. In the presence of X-Gal and IPTG, re-combinant phage plaques were colorless. The control was blue. Screening the library with GCF-5 McAb, apositive plaque had been isolated. In subsequent repeated cloning, all plaques were GCF-5 stained. Thepositive phages from the positive plaque were used to adsorb E. Coli. Y1089, and cultured scparately at 32℃and 42℃ . The lysogens, which grew confluently at 32℃ and spottily at 42℃, were cultured and induced toproduce fusion proteins with IPTG at 42℃. SDS-PAGE showed a slightly larger band than -galactosidase in thelysogens. GCF-5 McAb, after co-incubation with the crude lysate of the lysogens, could not stain the GCTcell, where as the GCF-5 McAb, incubated with crude lysate of control phage showed positive stain. Conclu-sions After a series of complex procedure, a whole cDNA library of GCT was constructed. GCF-5 antigen'scDNA was cloned out by GCF-5 McAb. The fusion protein contain the GCF-5 antigen were produced by in-duction with IPTG and proper temperature. Cyto-immunology block assay confirmed all of the result.