Lotfi H, Hejazi M A, Heshmati M K, Mohammadi S A, Zarghami N
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Genomics, Branch for the Northwest and West Region, Agricultural Biotechnology Research Institute of Iran (ABRII), Agriculture research, Education and Extension Organization (AREEO), Tabriz, Iran.
Cell Mol Biol (Noisy-le-grand). 2017 Sep 30;63(9):96-105. doi: 10.14715/cmb/2017.63.9.17.
Cyanovirin-N (CVN) is well known as an anti-HIV protein. The efficient production of low cost microbicides for preventing the HIV-infection has lately become a requirement worldwide. The aim of the present study was to optimize the expression of antiviral Cyanovirin-N homology gene found in the indigenous strain of Nostoc ellipsospourum LZN using Response Surface Methodology (RSM) and Protein Structure Analysis. Optimization of three induction factors (IPTG concentration (0.1, 0.55 and 1mM), temperature for bacterial growth (20, 28.5 and 37°C) and induction time (4, 10 and 16h) was done using RSM and Box-Behnken Design. Total RNA extraction was performed and mRNA levels were quantified in each experimental design by one-step SYBR qPCR. Protein structure was predicted using I-TASSER server. The full-length sequence of LZN-CVN gene is 306 bp in length, due mostly to five mutations. RSM analysis showed that the optimum condition to obtain maximum fold change was a concentration of 0.6mM IPTG, temperature set to 29°C and a 12h long induction time. The extracted protein from periplasmic fraction (8 kDa) was verified via SDS-PAGE. The high percentage of LZN-CVN similarity was demonstrated with PDB (Protein Data Bank) accession code of 2rp3A (CVN domain B mutant) and the ligand binding sites were related to N42, V43, D44, G45, S52, N53 and E56 residues. Different expression systems could assist in the development of anti-HIV proteins in a large scale. The LZN-CVN protein was successfully expressed in the E.coli system. RSM could be applied to a series of mathematical and statistical methods for modeling and analysis of responses which are influenced by various variables of interest.
氰病毒素 - N（CVN）是一种著名的抗HIV蛋白。高效生产低成本的用于预防HIV感染的杀菌剂近来已成为全球的一项需求。本研究的目的是利用响应面法（RSM）和蛋白质结构分析优化在椭圆念珠藻LZN本地菌株中发现的抗病毒氰病毒素 - N同源基因的表达。使用RSM和Box - Behnken设计对三个诱导因子（IPTG浓度（0.1、0.55和1mM）、细菌生长温度（20、28.5和37°C）以及诱导时间（4、10和16小时））进行优化。进行总RNA提取，并通过一步SYBR qPCR在每个实验设计中对mRNA水平进行定量。使用I - TASSER服务器预测蛋白质结构。LZN - CVN基因的全长序列为306 bp，主要是由于五个突变。RSM分析表明，获得最大倍数变化的最佳条件是IPTG浓度为0.6mM、温度设定为29°C以及诱导时间为12小时。通过SDS - PAGE验证了从周质部分提取的8 kDa蛋白质。LZN - CVN与蛋白质数据库（PDB）登录号为2rp3A（CVN结构域B突变体）具有较高的相似性，并且配体结合位点与N42、V43、D44、G45、S52、N53和E56残基相关。不同的表达系统有助于大规模开发抗HIV蛋白。LZN - CVN蛋白在大肠杆菌系统中成功表达。RSM可应用于一系列数学和统计方法，用于对受各种感兴趣变量影响的响应进行建模和分析。
