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根据NCBI数据库中的黄曲霉细胞壁蛋白AFLMP1的基因序列设计特异引物,以黄曲霉菌总RNA逆转录获得的c DNA为模板,PCR扩增目的基因后分别克隆到p GEX-6p-1和p ET-22b原核表达载体中,转化大肠杆菌BL21(DE3)感受态细胞。重组大肠杆菌经IPTG诱导表达后超声波破碎提取可溶性蛋白,SDS-PAGE电泳和Western杂交检测表达产物,结果表明GST-AFLMP1和AFLMP1-His融合蛋白能够在大肠杆菌中大量可溶性表达,可分别作为制备AFLMP1特异抗体的免疫抗原和筛选抗原。
Specific primers were designed according to the gene sequence of aflatoxin cell wall protein AFLMP1 in the NCBI database. The cDNAs obtained by reverse transcription of total RNA of Aspergillus flavus were used as templates. After PCR amplification of the target genes, they were cloned into pGEX-6p-1 and p ET -22b prokaryotic expression vector, transformed into E. coli BL21 (DE3) competent cells. The recombinant E.coli was induced by IPTG and then the soluble proteins were extracted by sonication. The expressed products were detected by SDS-PAGE and Western blotting. The results showed that GST-AFLMP1 and AFLMP1-His fusion proteins could be expressed in E. coli in large quantities and could be used as the preparation of AFLMP1 Antibodies to specific antibodies and screening antigens.