CAJ | 학술논문

目的探讨表达不同氨基端二级结构的丙型肝炎病毒非结构蛋白3/4A(HCV NS3/4A)各亚型丝氨酸酶活性及其对宿主细胞功能抑制作用的差异.方法以pSG5/M-H05-5/4A为模板(A1-1),构建表达不同氨基端二级结构NS3/4A的点突变质粒,并分别命名为A1-2,A2-1,A2.2,B1-1,B1-2,B2-1,B2-2.Western印迹检测所有构建质粒的表达及各亚型顺式及反式NS3蛋白酶活性的差异.荧光素酶报告试验检测pSG5/M-H05-5/4A各个二级结构亚型对干扰素-β(IFN-β)产生及p53依赖的荧光素酶基因的转录活性的抑制作用以及各亚型之间抑制作用的差异.结果 Western印迹显示所有构建质粒均成功表达,而且A2-1和B2-1亚型NS3/4A存在不完全切割现象.表明与其他亚型相比,A2-1和B2-1顺式NS3丝氨酸蛋白酶活性较弱.与其底物NS5A/5BAC共表达后,A2-1和B2-1亚型未切割NS5A5B明显多于其他亚型,而切割 NS5A 则明显少于其他亚型.说明与其他亚型相比,A2-1和B2-1 亚型的反式NS3蛋白酶活性亦较弱.荧光素酶试验结果显示,所有亚型M-H05-5/4A均显著抑制IFN-β启动子活性(P＜0.01)和p53依赖的荧光素酶基因的转录活性(P＜0.01),A2-1和B2-1亚型的抑制作用显著弱于其他亚型(P＜0.05).结论氨基端不同二级结构的HCV NS3/4A复合体具有不同的丝氨酸酶活性和宿主细胞功能抑制作用.
목적 탐토표체불동안기단이급결구적병형간염병독비결구단백3/4A(HCV NS3/4A)각아형사안산매활성급기대숙주세포공능억제작용적차이.방법 이pSG5/M-H05-5/4A위모판(A1-1),구건표체불동안기단이급결구NS3/4A적점돌변질립,병분별명명위A1-2,A2-1,A2.2,B1-1,B1-2,B2-1,B2-2.Western인적검측소유구건질립적표체급각아형순식급반식NS3단백매활성적차이.형광소매보고시험검측pSG5/M-H05-5/4A각개이급결구아형대간우소-β(IFN-β)산생급p53의뢰적형광소매기인적전록활성적억제작용이급각아형지간억제작용적차이.결과 Western인적현시소유구건질립균성공표체,이차A2-1화B2-1아형NS3/4A존재불완전절할현상.표명여기타아형상비,A2-1화B2-1순식NS3사안산단백매활성교약.여기저물NS5A/5BAC공표체후,A2-1화B2-1아형미절할NS5A5B명현다우기타아형,이절할 NS5A 칙명현소우기타아형.설명여기타아형상비,A2-1화B2-1 아형적반식NS3단백매활성역교약.형광소매시험결과현시,소유아형M-H05-5/4A균현저억제IFN-β계동자활성(P＜0.01)화p53의뢰적형광소매기인적전록활성(P＜0.01),A2-1화B2-1아형적억제작용현저약우기타아형(P＜0.05).결론 안기단불동이급결구적HCV NS3/4A복합체구유불동적사안산매활성화숙주세포공능억제작용.
Objective To construct the point mutation plasmids expressing NS3/4A with different secondary structure of the amino-terminal 120 residues of NS3,and to investigate the differenees in serine protease and inhibitory effects on host cells between each subgroup.Methods The point mutation plasmids were constructed,which expressed NS3/4A with the corresponding secondary structures of subgroup,and were named as A1-2,A2-1,A2-2,B1-1,B1-2,B2-1,and B2-2,with the backbone of M-H05-5(A1-1).Western blot was performed to detect the expression of NS3/4A and the difference in in cis and in trans NS3 serine protease activity between each subgroup.The inhibitory effects of HCV NS3/4A with different aminoterminal secondary structures on IFN-β production and p53-dependent transcriptional activation were revealed by Luciferase reporter assay.Results Western blot revealed the successful expression of the constructs and the incomplete cleavage of NS3/4A in subgroup A2-1 and B2-1.indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker compared with that of the other subgroups.By using NS5A/5B△C as a substrate for NS3/4A serine protease,it was also found that the in trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared with that of the other subgroups.Differences in inhibitory effects of HCV NS3 on IFN-β promoter activity and on p53-dependent lucifevase gene transcriptional activation were also observed between subgroup A2-1,B2-1 and the other subgroups.Conclusion HCV NS3/4A with different secondary structures at amino-terminus has different serine protease activities and inhibitory activities on host cell functlons.