目的 构建包含HCV核心蛋白全长基因的重组原核表达质粒,经转化表达型大肠杆菌后自诱导培养以获取可溶性重组核心蛋白,并检测其生物学功能. 方法 以H/FL质粒为模板,通过PCR扩增全长HCV核心蛋白DNA.PCR产物经双酶切后定向克隆到pET28a原核表达载体中.将重组的原核表达载体转化表达型大肠杆菌BL21 (DE3) pLysS,并通过自诱导培养的方式使其在高浓度状态下表达重组核心蛋白.重组核心蛋白经Western blot检测鉴定生物学功能,并与HCV NS3蛋白进行相互结合后共同纯化实验. 结果 重组核心蛋白大量存在于表达菌破菌上清液中.Western blot检测结果显示其具有核心蛋白抗原性.重组核心蛋白不能单独通过Ni-NTA亲和层析柱纯化,但可以与HCV NS3蛋白结合后共同纯化. 结论 成功表达可溶性的重组核心蛋白,证明了其生物学活性.重组核心蛋白与HCV NS3蛋白存在相互作用.

Objective To investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro ceil-based system.Methods The PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector.The recombinant plasmid was transformed into BL21 (DE3)pLysS E.coli to achieve high-concentration expression of the recombinant C protein by auto-induction.The recombinant protein C was purified by NiNTA affinity chromatography,and tested in a protein binding assay for its ability to bind the HCV NS3 protein.Results The transformed E.coli produced a large amount of recombinant protein C,as detected in the sonicated supernatant of the bacteria culture.The antigenic reactivity of the recombinant protein C was confirmed by western blotting.However,the recombinant protein C could not be purified by Ni-NTA affinity chromatography,but co-precipitated with the HCV NS3 protein.Conclusion S

目的 探讨丙型肝炎病毒(HCV)核心蛋白对双链RNA依赖的蛋白激酶(PKR)活性的影响. 方法 将表达HCV核心蛋白的真核表达质粒pCMH6K-Core转染人肝癌细胞系BEL-7402,应用干扰素(IFN) α-2b诱导内源性PKR的表达及活化,利用Western blot检测核心蛋白对PKR磷酸化的影响;将荧光素酶质粒pGL3-Promoter与不同剂量的pCMH6K-Core共转染BEL-7402细胞,进行荧光素酶活性检测,以反映核心蛋白对细胞蛋白合成的影响.计量资料以均数±标准差((x)±s)表示,组间均数比较用t检验或单因素方差分析. 结果 在IFNα-2b刺激下,表达HCV核心蛋白的BEL-7402细胞中,PKR的磷酸化水平明显低于空白质粒转染组和未转染组,而3组总PKR的表达水平无明显差别.在共转染荧光素酶质粒与核心蛋白表达质粒的BEL-7402细胞中,荧光素酶活性较共转染空白质粒组增强,且荧光素酶活性的增高与核心蛋白的表达量呈剂量依赖效应,0.5 μg、1.0μg和1.5μg的pCMH6K-Core转染组荧光素酶活性分别为空白质粒组的(1.941±0.199)倍、(2.868±0.275)倍和(3.839±0.338)倍,各组比较,P值均＜0.05. 结论 在人肝癌细胞系BEL-7402中,HCV核心蛋白能够抑制内源性PKR的活性,从而促进细胞蛋白合成.

Objective To explore the effects of hepatitis C virus (HCV) core protein on the activity of double-stranded RNA-dependent protein kinase (PKR).Methods The human hepatoma cell line BEL-7402 was transfected with the HCV core gene-containing eukaryotic expression vector pCMH6K-Core (at various concentrations),or empty vector,or no vector; a group of cells was co-transfected with the luciferase reporter plasmid pGL3-promoter.The cells were treated with interferon (IFN) α-2b to induce the expression and activation of endogenous PKR,or left untreated to serve as controls.The effect of core protein on PKR phosphorylation was detected by western blotting.Luciferase activity was detected to reflect effects of the core protein on the synthesis of cellular proteins.The t-test and F test were used for statistical analyses.Results In the case of IFNα stimulation,PKR phosphorylation levels were significantly lower in the HCV core protein expressing cells than in the cells transfected wit

丙型肝炎病毒（HCV）结构蛋白包括核心蛋白和2个包膜糖蛋白，即包膜糖蛋白 E1和包膜糖蛋白 E2；非结构蛋白包括 NS1、NS2、NS3、NS4A、NS4B、NS5A 和 NS5B。结构蛋白主要在病毒颗粒侵入与组装中发挥作用。非结构蛋白与感染细胞蛋白相互作用，在病毒的复制以及病毒致病过程起关键作用。

Hepatitis C virus(HCV)structural protein is composed of core protein and two en-velope glycoproteins E1 and E2.The nonstructural proteins consist of NS2,NS3,NS4A,NS4B, NS5A and NS5B.Structural proteins are involved in the entry and assembly of viral particles.The nonstructural proteins interact with cellular proteins and participate in viral replication and patho-genesis.

肝细胞癌(HCC)的发生与乙型肝炎病毒(HBV)感染密切相关。HBV感染所致的炎症反应、HBV X基因及其产物X蛋白、核心启动子突变、前S2区突变、剪接特异性蛋白等均可能参与HCC的发生发展。

The infection of hepatitis B virus (HBV) plays an important role in the occurrence of hepatocellular carcinoma (HCC). Multiple factors may be involved in the hepatocarcinogenesis of HBV-related HCC,such as inflammatory response,HBV X gene and its protein,the mutation of HBV core promoter and pre-S 2 genes,HBV spliced protein and so on. We will discuss some progresses in this field.

细胞周期蛋白（Cyclins）是细胞周期调节的核心分子，与细胞周期蛋白依赖性激酶（CDKs）、细胞周期蛋白依赖性激酶抑制因子（CKIs）等组成细胞周期调控网络系统。细胞周期调控机制的异常与肿瘤的发生发展密切相关，细胞周期蛋白的异常表达与CDKs结合形成的复合物，将导致细胞周期紊乱、细胞增殖失控，最终促使多种细胞癌变。Cyclins在多种恶性肿瘤中高表达，并在肿瘤的治疗中发挥重要作用。

Cyclins are critical modulators of cell cycle regulation. The cell cycle regulation system is constituted by cyclins, cyclin-dependent protein kinases (CDKs) and cyclin-dependent kinases inhibitors (CKIs). Abnormal cell cycle regulation mechanism is closely related to tumor development and progression. Abnormal expression of cyclins and the compound of CDKs and cyclins will lead to cell cycle disorder, cell proliferation and canceration. Cyclins are highly expressed in a variety of malignant tumors, which play an important role in tumor treatment.