PcDNA4/His C-MBL表达载体的构建及其在CHO细胞中的表达

摘要/Abstract

摘要： 目的构建PcDNA4/HisC-MBL表达载体,在哺乳细胞中表达人甘露聚糖结合凝集素(MBL)。方法应用PCR从含有中国人野生型MBLcDNA的质粒pGEM-MBL中扩增目的基因片段,将其克隆至PcDNA4/HisC真核表达载体。经酶切及测序鉴定后,以电穿孔法转染CHO细胞,以RT-PCR分析其mRNA表达,通过Ni-NTA琼脂糖柱层析纯化目的蛋白并以SDS-PAGE和Western-blotting鉴定,以其为免疫原免疫小鼠后取鼠血清进行ELISA检测分析目的蛋白的免疫活性。结果从pGEM-MBL中扩增得到约750bp的cDNA片段,构建成重组载体经酶切出现约5100和750bp片段,测序鉴定与预期的完全一致。RT-PCR证实转染细胞有MBLmRNA表达,纯化蛋白经SDS-PAGE电泳可见约29000、58000和87000条带,其中29000蛋白带可与6-His抗体反应。纯化蛋白免疫小鼠所获抗血清ELISA效价是1:819200,与天然人MBL和重组MBL三聚体糖识别域反应的ELISA效价均为1:25600。结论获得了表达中国人MBL的CHO细胞株和重组人MBL,为MBL分子的进一步研究提供了条件。

Abstract: Objective To construct pcDNA4/His C-MBL recombinant eukaryotic expression plasmid and examine its expression of mannan-binding lectin (MBL) in mammary cells. Methods The target sequence was amplified by PCR from pGEM-MBL plasmid that contains wild-type human MBL cDNA, and inserted into eukaryotic expression vector PcDNA4/His C followed by restriction mapping and sequencing. The recombinant plasmid PcDNA4/His C-MBL was transformed into Chinese-hamster ovary (CHO) cells by electroporation, and the Zeocin-resistant clones were selected for analysis of mRNA expression by reverse transcription (RT)-PCR. The expressed product was purified by immobilized metal affinity chromatography (IMAC) and identified by SDS-PAGE and Western-blot analysis, and its immunoreactivity was detected by an indirect enzyme-linked immunosorbent assay(LISA)sing the anti-serum from Balb/C mice immunized with the recombinant protein. Results The cDNA fragment of 750 bp was amplified from pGEM-MBL plasmid, which was shown by restriction enzyme digestion and DNA sequencing. The mRNA expression of Zeocin-resistant CHO cell clones was detected by RT-PCR. Three components of 29, 58 and 87 kD in the purified recombinant product were found by SDS-PAGE and the 29 kD component could be recognized by anti-6His antibody in Western blot analysis. The titers of the anti-serum from immunized mice were 1: 819 200 against the recombinant protein and 1:25 600 against both the natural human MBL and the recombinant trimeric carbohydrate-recognition domain (CRD) of human MBL, as determined by the indirect ELISA. Conclusion The cell strains that express recombinant human MBL (rhMBL) and rhMBL protein have been obtained successfully, which provides the basis for further research of MBL molecule.

中图分类号:

R392.1

白志军, 张丽芸, 林佐贤, 卢晓, 陈政良. PcDNA4/His C-MBL表达载体的构建及其在CHO细胞中的表达[J]. 南方医科大学学报, 2004, 24(08): 859-863.

BAI Zhi-jun, ZHANG Li-yun, LIN Zuo-xian, LU Xiao, CHEN Zheng-liang. Construction of the expression vector PcDNA4/His C-MBL and its expression in Chinese- hamster ovary cells[J]. Journal of Southern Medical University, 2004, 24(08): 859-863.

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