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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。
OBJECTIVE: To explore the mechanism of MBT gene CGT52TGT point mutation in phagocytosis. Methods: MBL gene containing CGT52TGT point mutation was obtained from plasmid pMBLm52 by PCR and inserted into eukaryotic expression vector pcDNA4 / HisMaxC to construct a recombinant expression vector. After sequencing validation, electroporation into CHO cells. CHO cells transfected at 800 mg / L Zeocin were screened for 30 days; the concentration of Zeocin was maintained at 200 mg / L for the next 30 days to obtain stable transfected cells. Its mRNA expression was analyzed by RT PCR. After purification of the expressed product by Ni NTA agarose, the expressed products were preliminarily identified by non-reducing SDS PAGE and Western blot. Results: The fragment of MBLm52 amplified by PCR was about 750bp in length and inserted into the expression vector to construct the recombinant eukaryotic expression vector pcDNA4 / HisMaxC MBLm52. The correct sequence was sequenced and transfected into CHO cells. The purified expression product obtained from the cell culture supernatant was mainly composed of molecules with a molecular weight of about 60,000 and the degree of oligomerization was significantly lower than that of the recombinant human wild type MBL and the human MBL isolated from human plasma. CONCLUSION: The CGT52TGT point mutation in MBL gene may not affect the excretion of its expressed product. However, the Cys produced after mutation may form new disulfide bonds, which may affect the formation of MBL structural units and / or oligomers, suggesting that this mutation MBL protein can not play a normal function.