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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 GCF-5 is a GCT-specific monoclonal antibody, and its antigen is one of the ways to understand the characteristics of GCT. To utilize this approach, the cDNA of the GCF-5 antigen was cloned and expressed. Methods Total RNA was extracted with guanidine hydrochloride and organic solvent, mRNA was isolated by Oligo(dT)-Cellulose affinity chromatography, cDNA was synthesized by reverse transcription, cDNA library was constructed with gt11Sfi-Not expression vector, and immunological screening and cloning of GCF-5 antigen. The cDNA, E.coli Y1089 expresses a fusion protein containing the GCF-5 antigen, and the cell immunosuppression experiment confirmed that the fusion protein contains the GCF-5 antigen component. Results The mRNA isolated by Oligo(dT)-Cellulose affinity chromatography had a concentration of 20.56 μg/ml, an OD260/280 ratio of 1.869, and cDNA products obtained from reverse transcription ranged from hundreds to thousands of bp. The product was complete; the constructed gt11Sfi-Not vector was colorless and transparent plaque after packaging in vitro, and a positive plaque was screened out with antibodies. The plaques obtained after repeated cloning were stained by antibodies; this phage infects Escherichia coli Y1089 was cultured at 32°C and 42°C, lysogens were selected, and IPTG was used to induce the fusion protein. SDS-PAGE showed slightly larger fusion bands than the normal β-galactosidase and was made by preincubation with the fusion protein. Cell immunochemical staining does not show color. Conclusion A complete GCT cDNA library was constructed.