SIV p27单克隆抗体的制备和鉴定

Preparation and Characterization of the Monoclonal Antibody Against SIV p27 Antigen

目的建立SIVp27杂交瘤细胞株，并对其分泌的SIVp27单克隆抗体进行初步鉴定。方法使用基因重组的SIVp27蛋白免疫BALB/c小鼠，采用杂交瘤技术使用半固体培养基法建立杂交瘤细胞株，制备单克隆抗体。通过染色体核型对杂交瘤细胞株进行鉴定；采用Western blot、免疫荧光法、酶联免疫吸附法确定单克隆抗体的交叉反应性、相对亲和力、抗原识别表位、免疫球蛋白的类型和亚类。对单克隆抗体进行鉴定。结果获得4株可稳定分泌SIVp27单克隆抗体的杂交瘤细胞，IC3、286为IgG1类，2E12为IgG2b类，3G3为IgG2a类。4株单抗均能识别SIV的p27蛋白，与逆转录病毒SRV、STLV无交叉反应，286、2E12与H1Vp24有交叉反应。免疫荧光法检测腹水效价为1：10240～1：40960。1C3、286、2E12、3G3染色体平均数分别为103、97、96、101。2E12与3G3识别不同的抗原表位。结论成功地制备出4株SIVp27单克隆抗体，均具有良好的特异性和亲和力，为进一步建立免疫分析方法，进行SIV／SAIDS及其艾滋病相关研究，奠定了基础。

Objective （1） To establish SIV p27 hybridoma cell lines. （2） To perform elementary identification of monoclonal antibodies （McAb）. Methods BALB/c mice were immunized with recombinant SIV p27 protein. Hybridoma cell lines were set up by using hybridoma technique and semisolid culture medium and monoclonal antibodies were prepared. The hybridoma cell lines were verified by chromosome analysis. The monoclonal antibodies were identified by assessment of cross reactivity, relative affinity, antigenic epitopes they recognized and the immunoglobulin class determined by Western blot, immunofluorescence assay and enzyme linked immunoserbent assay （ ELISA）. Results Four hybfidoma cell lines that stably secreted SIV p27 McAbs were obtained： 1C3 and 2B6 belonging to IgG1,2E12 belonging to IgG2b, and 3G3 belonging to IgG2a. All four McAbs bound with SIV p27 protein, but had no cross-reactivity with SRV and STLV. However, 2B6 and 2EI2 had crossreactivity with HIV p24. The titers of aseites were 1 ： 10240 - 1：40960, showed by immunofluoreseenee assay. The averages of chromosome of IC3, 2B6, 2E12 and 3G3 were 103, 97, 96 and 101, respectively. Different antigenic epitopes were recognized by 2E12 and 3G3. Conclusions Four monoclonal antibodies against SIV p27 protein with high specificity and affinity have been successfully generated, which may provide a basis for further establishing immunological assay and for SIV/SAIDS and related performances in AIDS studies.