人CGT52TGT MBL突变体在CHO细胞中的表达及其产物分析

目的: 初步探索MBL基因CGT52TGT点突变引起调理吞噬缺损的机制。方法: 采用PCR技术, 从质粒pMBLm52中获取含CGT52TGT点突变的MBL基因, 将其插入真核表达载体pcDNA4 /HisMaxC中构建重组表达载体。经测序验证后, 电转染入CHO细胞。以800mg/LZeocin筛选转染后的CHO细胞30d; 随后的30d中, 维持Zeocin的浓度在200mg/L, 以获取稳定转染的细胞。以RT PCR分析其mRNA的表达情况。表达产物经Ni NTAagarose纯化后, 以非还原SDS PAGE和Westernblot对表达产物进行初步鉴定。结果:以PCR扩增的MBLm52基因片段长约750bp, 将其插入表达载体构建重组真核表达载体pcDNA4 /HisMaxC MBLm52, 测序验证序列正确后将其电转染入CHO细胞。从细胞培养上清中获得的纯化的表达产物, 主要为相对分子质量(Mr)约60 000的分子, 寡聚化程度明显低于重组人野生型MBL和从人血浆中分离的MBL。结论: MBL基因CGT52TGT点突变可能并不影响其表达产物向胞外分泌的过程, 但突变后产生的Cys可能形成新的二硫键, 影响MBL结构单位和/或寡聚分子的形成, 推测该突变MBL蛋白不能发挥正常的功能。

Expression of human CGT52TGT MBL mutant in CHO cells and analysis of the expression product

AIM: To explore preliminarily the mechanisms of immunodeficiency resulted from the CGT52TGT point mutation of mannan-binding lectin(MBL) gene. METHODS: The MBL gene containing CGT52TGT point mutation was amplified from the plasmid pMBLm52 by PCR, and then inserted into the eukaryotic expression vector pcDNA4/HisMax C. After confirmed by DNA sequencing, the recombinant expression vector was transfected into Chinese-hamster ovary(CHO) cells by electroporation. Zeocin of 800 mg/L had been used for 30 days to select electroporated CHO cells, and then 200 mg/L for another 30 days to obtain stable transfectant. The expression of mRNA was analyzed by RT-PCR, the recombinant protein was purified from the culture supernatant by Ni-NTA agarose chromatography and analyzed by SDS-PAGE under nonreducing condition and Western blot. RESULTS: The cDNA fragment amplified from pMBLm52 plasmid by PCR was about 750 bp and the recombinant plasmid pcDNA4/HisMax C-MBLm52 was constructed and transfected into CHO cells. The expression product purified from the culture supernatant appeared mainly at the site of M(r) 60,000, indicating a much lower oligomerization level than that of the recombinant human wild MBL and human plasma-derived MBL. CONCLUSION: The CGT52TGT point mutation of MBL gene does not affect the secretion of its product, but a Cys introduced by the mutation could form another disulfide bond which may disrupt the structure of MBL molecule as well as its function.