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首页> 外文期刊>African Journal of Biotechnology >Genomic sequencing and recombinant expression of proinsulin isolated from cow and buffalo in Pakistan
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Genomic sequencing and recombinant expression of proinsulin isolated from cow and buffalo in Pakistan

机译：巴基斯坦牛和水牛中胰岛素原的基因组测序和重组表达

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摘要

This study was conducted to determine the heterologous expression of eukaryotic gene in Escherichia coli variable. The expression of Pakistani buffalo (Bubalus bubalis) and cow (Bos taurus) proinsulin genes in BL21 codon plus cells is 30% of total cellular protein. Total RNA was isolated from pancreatic tissues of local (Pakistani) breeds of buffalo (B. bubalis) and cow (B. taurus), converted to cDNA and cloned in a T/A cloning vector.? There are five silent mutations: one in B-chain, two each in A-chain and C-peptide encoding regions of local Pakistani buffalo proinsulin DNA sequence as compared to the internationally reported cow proinsulin sequence. The DNA sequence of local (Pakistani) cow proinsulin shows there is one nucleotide encodes C-peptide that has mismatch with the reported cow proinsulin sequence but mismatched nucleotide is same between Pakistani cow and Pakistani buffalo proinsulin sequence. This indicates the genetic similarity of Pakistani cow with Pakistani buffalo.? Both genes were expressed in BL21 Codon plus (DE3)-RIL cells with 0.2 mM IPTG at 37°C for 6 to 8 h. Proinsulin was expressed as 30% of total cellular proteins as insoluble inclusion bodies.
机译：进行这项研究以确定真核基因在大肠杆菌变量中的异源表达。巴基斯坦水牛（Bubalus bubalis）和牛（Bos taurus）前胰岛素基因在BL21密码子加细胞中的表达占总细胞蛋白的30％。从水牛（B. bubalis）和牛（B. taurus）的本地（Pakistani）品种的胰腺组织中分离总RNA，将其转化为cDNA，并克隆到T / A克隆载体中。与国际上报道的牛胰岛素原序列相比，有五个沉默突变：一个在巴基斯坦本地水牛胰岛素原DNA序列的B链，两个在A链和C肽编码区。本地（巴基斯坦）牛胰岛素原的DNA序列显示，有一个编码C肽的核苷酸与报道的牛胰岛素原序列不匹配，但巴基斯坦牛和巴基斯坦水牛胰岛素原序列之间的错配核苷酸相同。这表明巴基斯坦母牛与巴基斯坦水牛的遗传相似性。两种基因均在具有0.2 mM IPTG的BL21密码子加（DE3）-RIL细胞中于37°C表达6至8小时。胰岛素原表达为不溶性包涵体占全部细胞蛋白的30％。

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