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VP22融合型顯性負(fù)性突變體抑制乙肝病毒復(fù)制
作者：易軍，宮衛(wèi)東，王嶺，凌瑞，陳江浩，王輝，王廷
【關(guān)鍵詞】 肝炎病毒
Inhibition of hepatitis B virus replication by dominant negative mutant of VP22 fusion protein
【Abstract】 AIM: To investigate the inhibitory effect on hepatitis B virus (HBV) replication with dominant negative (DN) mutant of VP22 fusion protein. METHODS: Fulllength or fractions of VP22 were fused to C terminal of HBV core protein (HBc), and cloned into pcDNA3.1(-) vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the concentration of HBV surface antigen (HBsAg) in the supernatant of cell culture. And MTT assay was performed to detect the cytotoxicity of transgene expression to the host cells. RESULTS: DN mutant of VP22 and its fractions could be expressed in HepG2.2.15 cells, and had no toxic effect on the host cells. The DN mutant could inhibit HBV replication, and the mutant with protein transduction ability had a stronger inhibition than that without. The DN mutant of fulllength VP22 had the strongest inhibitory effect, reducing the HBsAg concentration by 81.94%. CONCLUSION: DN mutant of VP22 fusion protein can enhance the inhibition of HBV replication.
【Keywords】hepatitis B virus; dominant negative mutant; VP22
【摘要】 目的： 了解VP22融合型顯性負(fù)性(DN)突變體對(duì)抑制乙肝病毒(HBV)復(fù)制的作用. 方法： 將VP22全長(zhǎng)及其不同區(qū)段融合于HBV核心蛋白(HBc)的C端，克隆入pcDNA3.1(-)構(gòu)成DN突變體真核表達(dá)質(zhì)粒. 轉(zhuǎn)染HepG2.2.15細(xì)胞后，免疫熒光鑒定DN突變體在細(xì)胞內(nèi)的表達(dá)，并以培養(yǎng)上清HBV表面抗原(HBsAg)的濃度為指標(biāo)，觀察DN突變體對(duì)HBV病毒復(fù)制的抑制效應(yīng). 同時(shí)以MTT比色法觀察轉(zhuǎn)基因的表達(dá)對(duì)宿主細(xì)胞生長(zhǎng)狀態(tài)的影響. 結(jié)果： VP22及其不同區(qū)段構(gòu)建的DN突變體可在HepG2.2.15細(xì)胞中進(jìn)行表達(dá)，且對(duì)宿主細(xì)胞無毒性. VP22融合型DN突變體可有效地抑制HBV的復(fù)制，具有蛋白轉(zhuǎn)導(dǎo)特性的DN突變體比無轉(zhuǎn)導(dǎo)特性的DN突變體具有更強(qiáng)的抗病毒能力. 其中VP22全長(zhǎng)構(gòu)成的DN突變體具有最強(qiáng)的抑制效應(yīng)，可使上清HBsAg濃度下降81.94%. 結(jié)論： VP22融合型DN突變體可以增強(qiáng)DN突變體對(duì)HBV復(fù)制的抑制.
【關(guān)鍵詞】 肝炎病毒，乙型；顯性負(fù)性突變體；VP22
0引言
全球目前有3.5億慢性乙型肝炎病毒（hepatitis virus B, HBV）感染者，每年約有100萬人死于HBV感染的相關(guān)疾病，占疾病死因的第9位［1］. 目前，國(guó)際上尚無有效治療慢性乙肝的手段. 基因治療技術(shù)的不斷發(fā)展為抗HBV治療提供了新的思路和途徑. 其中顯性負(fù)性(dominant negative，DN)突變體的胞內(nèi)表達(dá)是HBV基因治療的重要策略之一［2-6］. Elliott等［7］發(fā)現(xiàn)，單純皰疹病毒1(herpes simplex virus type 1, HSV1)的結(jié)構(gòu)蛋白VP22(及其融合蛋白)可從培養(yǎng)基進(jìn)入細(xì)胞內(nèi)，并可不經(jīng)過細(xì)胞細(xì)胞接觸依賴性在同型或異型細(xì)胞之間轉(zhuǎn)移. 我們將HBV核心蛋白(HBV core protein, HBc)與VP22進(jìn)行融合構(gòu)成DN突變體，以期利用VP22的蛋白轉(zhuǎn)導(dǎo)特性進(jìn)一步提高DN突變體的抗病毒效應(yīng).
1材料和方法
1.1材料質(zhì)粒pVP22/mycHis2和pcDNA3.1(-)為Invitrogen公司產(chǎn)品；EBOHBV克隆有1.3倍HBV全長(zhǎng)基因組為本室保存. 限制性內(nèi)切酶、連接酶購(gòu)自TaKaRa Biotech Co.Ltd；鼠抗HBc抗體和鼠抗cmyc抗體為SantaCruz公司產(chǎn)品，F(xiàn)ITC標(biāo)記的羊抗鼠IgG為博士德生物工程有限公司產(chǎn)品；DMEM培養(yǎng)基、進(jìn)
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