摘要
本文研究了野生和突变H7N9禽流感病毒神经氨酸酶（Neuraminidase,NA）对奥司他韦羧酸盐及扎那米韦的敏感性。将密码子优化的H7N9（A/Hangzhou/1/2013）病毒神经氨酸酶DNA克隆到pcDNA3.1/His载体（简称NAH7N9-WT质粒）后,将该质粒转染293T细胞,48h后裂解收集上清液,得到野生型H7N9神经氨酸酶。利用一步PCR突变法,在NAH7N9-WT质粒中引入H274Y和R292K突变（NA以N2编号）,简称NAH7N9-H274Y、NAH7N9-R292K质粒,测序确认正确后将两种突变质粒转染293T细胞,48h后裂解收集上清液,获得突变H7N9神经氨酸酶。Western blot鉴定野生和突变NA的表达。以4-MUNANA为底物检测野生及突变NA活性,检测奥司他韦羧酸盐和扎那米韦对野生及突变NA的抑制活性。结果显示,野生及突变NA质粒转染细胞后均获得了分子量约为70kD的目标蛋白,但H274Y突变酶的表达量显著低于野生酶及R292K突变酶;所表达的NAH7N9-WT、NAH7N9-H274Y和NAH7N9-R292K均有活性;奥司他韦羧酸盐对NAH7N9-WT、NAH7N9-H274Y和NAH7N9-R292K的半数抑制浓度分别为1.6nM、15.1nM和大于1 000nM,耐药倍数分别为9和大于625倍;扎那米韦对NAH7N9-WT、NAH7N9-H274Y和NAH7N9-R292K的半数抑制浓度分别为1.1nM、1.4nM和38.0nM,耐药倍数分别为1.3和34倍。本研究结果表明奥司他韦和扎那米韦可显著抑制NAH7N9-WT活性,NAH7N9-R292K对奥司他韦和扎那米韦有显著的耐药性（耐药倍数34~625倍）,NAH7N9-H274Y对奥司他韦和扎那米韦敏感（耐药倍数1~9倍）。结果提示,临床感染H7N9的患者可用扎那米韦或奥司他韦治疗,但当该病毒发生NAH7N9-R292K突变时,建议停止使用这两种药物。
This study aimed to investigate the drug susceptibility of wild-type and mutant avian influenza A（H7N9）virus neuraminidase（NA）to oseltamivir and zanamivir.Codon optimized DNA of H7N9（A/Hangzhou/1/2013）NA was synthesized and constructed into the pcDNA3.1/His vector（NAH7N9-WT）.Mutant NAH7N9-H274Yand NAH7N9-R292Kplasmids were constructed by directed mutagenesis PCR using NAH7N9-WT plasmid as the template followed by sequencing.NA plasmids were transfected into 293Tcells and cell lysates containing NAs were collected 48hpost-transfection.Wild-type and mutant NAs were analyzed by Western blotting and their activities were tested by the 4-MUNANA-based assay.All three NAs were expressed and enzymatic activities were confirmed.The effects of oseltamivir and zanamivir on all three NAs were then tested.It showed that the half maximal inhibitory concentrations（IC50s）of oseltamivir carboxylate on NAH7N9-WT,NAH7N9-H274Yand NAH7N9-R292K were 1.6nM,15.1nM,and＆gt;1 000nM with fold changes of 9and＆gt;625,respectively.The IC50values of zanamivir on NAH7N9-WT,NAH7N9-H274Y,and NAH7N9-R292K were 1.1nM,1.4nM,and 38.0nM with fold changes of 1.3and 34,respectively.These results indicated that oseltamivir and zanamivir could significantly inhibit NAH7N9-WT.NAH7N9-R292Kshowed high-level resistance to both drugs（34-fold and 625-fold）and NAH7N9-H274Ywas sensitive to both（1.3-fold and 9-fold）.These results indicated that both oseltamivir and zanamivir could be used for patients infected with the H7N9virus.However,when patients carried the H7N9virus with a NA R292Kmutation,other medications would be preferred over oseltamivir or zanamivir.
出处
《病毒学报》
CAS
CSCD
北大核心
2014年第4期396-401,共6页
Chinese Journal of Virology
基金
国家自然科学基金青年基金(No.81202568)
高等学校博士学科点专项科研基金(20111106120022)
