论文部分内容阅读
免疫球蛋白可变区信息包含了Ｂ淋巴细胞克隆产生的分子机制，对了解抗原、抗体间相互作用及在自身免疫中的作用机制具有重要意义。本研究应用两对通用引物，采用细胞内ＰＣＲ，从分泌高亲和力抗人Ｃ１ｑ单抗杂交瘤细胞扩增其轻、重链可变区基因。即杂交瘤细胞以４％多聚甲醛固定，经ＮＰ４０处理增加膜通透性后，直接作细胞内反转录合成ｃＤＮＡ，继以ｃＤＮＡ为模板作细胞内ＰＣＲ，从而无需提取细胞ＲＮＡ或ＤＮＡ便扩增获得轻、重链Ｖ基因。同时，通过建立快速、高效、高分辨率毛细管电泳技术检测ＰＣＲ扩增产物，达到ＰＣＲ扩增及检测的自动化。扩增的轻、重链可变区基因分别克隆进ｐＧＥＭ－１１Ｚｆ（－）并作序列测定。计算机序列分析及同源性检索证实所克隆基因片段归属鼠免疫球蛋白家族。按Ｋａｂａｔ分类标准，抗－Ｃ１ｑ轻链Ｖ基因（Ｖκ）属鼠Ｉｇ第Ｖ组，而其重链Ｖ基因（ＶＨ）属ⅡＢ组。令人感兴趣的是，抗－Ｃ１ｑＶκ与自身抗体的Ｖκ高度同源，而抗－Ｃ１ｑＶＨ主要与胚系基因及某些自身抗体显著同源（达７５％以上）。由胚系基因编码的不同特异性抗体往往具有与自身抗原结合特性，自身抗体亦多由胚系基因编码。因此，本株高亲和力抗－Ｃ１ｑＩｇＧ与胚系基因及自身抗体Ｖ基因密切相?
Immunoglobulin variable region information contains the molecular mechanism of B lymphocyte cloning, and is of great significance to understand the mechanism of antigen, antibody interaction and autoimmunity. In this study, two pairs of universal primers were used to amplify the light and heavy chain variable region genes from secreting high affinity anti-human C1q monoclonal antibody hybridoma cells by intracellular PCR. That is, the hybridoma cells were fixed with 4% paraformaldehyde. After the permeability of the membrane was increased by NP40 treatment, the cDNAs were reverse transcribed directly into cDNAs, followed by cDNA as a template for intracellular PCR, thereby eliminating the need to extract cellular RNA or DNA Amplified light and heavy chain V gene. At the same time, PCR amplification products are detected by establishing rapid, efficient and high-resolution capillary electrophoresis to achieve the automation of PCR amplification and detection. The amplified light and heavy chain variable region genes were cloned into pGEM-11Zf (-) and sequenced. Computer sequence analysis and homology search confirmed that the cloned gene fragment belongs to murine immunoglobulin family. According to the Kabat classification, the anti-C1q light chain V gene (VK) is a mouse Ig group V and the heavy chain V gene (VH) is a group IIB. Interestingly, anti-C1qVκ is highly homologous to the Vκ of autoantibodies, whereas anti-C1qVH is predominantly homologous (up to 75%) with germline genes and some autoantibodies. Different germline genes encoded by different specific antibodies tend to have self-antigen binding properties, autoantibodies are also mostly encoded by germline genes. Therefore, the plant high affinity anti-C1qIgG germline genes and autoantibodies V gene close phase?