抗Ⅳ型胶原酶抗体Fv片段与力达霉素融合蛋白的免疫活性  

Immunoreactivity of Fusion Protein Fv-LDP Consisting of Lidamycin Apoprotein and the Fv fragment from anti-type Ⅳ Collagenase Antibody

作　　者：蔡琳[1,2] 郭效忠[3] 高瑞娟[1] 李亮[1] 甄永苏[1] 

机构地区：[1]中国医学科学院/北京协和医学院医药生物技术研究所,100050 [2]温州医学院药学院,325035 [3]河北省沧州市中心医院病理科,061000

出　　处：《医学研究杂志》2008年第11期35-39,143,共6页Journal of Medical Research

基　　金：国家自然科学基金资助(30701029)

摘　　要：目的融合蛋白Fv-LDP是通过基因工程方法制备,其抗体部分为抗Ⅳ型胶原酶抗体3G11的单链抗体scFv,相当于"弹头"的载体部分为力达霉素辅基蛋白LDP。本实验研究了融合蛋白Fv-LDP的免疫学活性。方法采用间接ELISA方法和免疫细胞荧光方法检测融合蛋白Fv-LDP的免疫学活性,明胶酶谱实验观察融合蛋白Fv-LDP对肿瘤细胞分泌明胶酶的影响,重组基膜侵袭实验分析融合蛋白Fv-LDP对肿瘤细胞侵袭能力的影响,通过制备兔抗Fv-LDP多抗,应用免疫组织化学技术检测融合蛋白Fv-LDP与人乳腺癌组织的免疫反应性。结果融合蛋白Fv-LDP与Ⅳ型胶原酶、纤维肉瘤HT-1080细胞、结肠癌HT-29细胞、肺癌PG细胞等均具有免疫反应性,3种细胞中以HT-1080细胞的相对亲和力最高,其次是PG细胞,HT-29细胞。免疫细胞荧光结果显示,肿瘤细胞HT-1080胞质着绿色荧光,表明融合蛋白Fv-LDP结合于肿瘤细胞的胞质部位。融合蛋白Fv-LDP可有效抑制肿瘤细胞分泌明胶酶,可明显抑制肿瘤细胞对人工基膜的侵袭,并呈剂量依赖性。人乳腺癌组织免疫组化结果显示,融合蛋白Fv-LDP与乳腺癌癌细胞胞质部位结合,阳性着色部位与MMP-9抗体的染色一致。结论融合蛋白Fv-LDP基本保留了完整单抗3G11对明胶酶的免疫活性,又因其携带有"弹头"的载体蛋白LDP,可以用烯二炔发色团进行强化,因此,有可能用于制备抗肿瘤抗体药物。Objective Fv fragment of 3G11 ,a murine monoclonal antibody directed against type Ⅳ collagenase,was linked to LDP, an apoprotein of lidamycin,to form fusion protein Fv - LDP. This study focuses on the immunoreactivity of the fusion protein Fv - LDP. Methods Indirect ELISA and immunofluorescent staining were used to detect the immunoreactivity of fusion protein. Gelatin zymography analysis was performed to observe the effect of fusion protein Fv - LDP on HT - 1080 cells. Reconstituted basement membrane invasion was conducted to examine the effect of fusion protein Fv - LDP on the invasion of tumor cells. Rabbit anti - Fv - LDP polyclonal antibody was prepared to evaluate the immunoreactivity of Fv - LDP with human breast carcinoma tissue by immunohistochemistry. Results Indirect ELISA showed the existence of immunoreactivity between fusion protein Fv - LDP and type Ⅳ collagenase, HT -29 cells, HT - 1080 cells and PG cells. HT - 1080 cells showed a higher relative affinity to fusion protein Fv - LDP than HT -29 cells and PG cells. Immunocytological staining showed that fusion protein Fv - LDP was positive to HT - 1080 cells and displayed green fluorescence in cytoplasm of the tumor cells. Type Ⅳ collagenase（ MMP - 2 and MMP - 9 ） activity was inhibited as shown by gelatin zymography. Fusion protein Fv - LDP inhibited the invasion of tumor cells upon basement membrane. Immunohistochemical staining of breast carcinoma tissue with fusion protein Fv - LDP indicated similar positive staining as anti - MMP - 9 antibody in carcinoma ceils. Concuision The results demonstrate that fu- sion protein Fv - LDP basically retains the immunoreactivity of mAb 3Gll to type Ⅳ collagenase and to relevant cancer cells. Therefore, fusion protein Fv - LDP may be used for preparation of antibody - based therapeutics.

关 键 词：Ⅳ型胶原酶 免疫活性 融合蛋白 基因工程 

分 类 号：R735.202[医药卫生—肿瘤] R285.5[医药卫生—临床医学]