携带反义凝血酶受体和p21双基因共表达腺病毒伴随病毒载体的构建与包装  

Construction and Package of Antisense Thrombin Receptor and p^(21) Co-expression Adeno-associated Virus Vector

作　　者：孟宪敏[1] 吴小兵[2] 米立国[1] 伍志坚[2] 赵秀文[1] 刘冬青[1] 丁金凤[1] 

机构地区：[1]中国医学科学院中国协和医科大学心血管病研究所,阜外心血管病医院分子医学中心,北京100037 [2]中国预防医学科学院病毒学研究所,基因工程国家重点实验室,北京100052

出　　处：《生物化学与生物物理进展》2002年第4期597-603,共7页Progress In Biochemistry and Biophysics

基　　金：国家"九五"攻关项目 (96 90 6 0 2 0 7) ;国家自然科学基金资助项目 (3 9970 2 97) ;国家"863"基因治疗重大课题 (863 BH0 3 0 5 0 2 )

摘　　要：为探讨双基因共表达对再狭窄的防治作用 ,分别构建了含反义凝血酶受体 (ATR)或 /和 p2 1单、双基因及报告基因绿色荧光蛋白 (GFP)的腺病毒伴随病毒 (AAV)载体 .上述载体经脂质体介导转染BHK 2 1细胞 ,G4 18筛选获得整合有外源基因的细胞株 .克隆形成过程中显示单、双基因转染后细胞增殖受到了不同程度的抑制 ,克隆形成速度减慢 ,细胞形态改变 ,且双基因的作用明显大于单基因 .以绿色荧光出现说明报告基因得到表达后 ,又以DNA印迹证实ATR和p2 1单、双基因已整合于细胞基因组中 ,并维持了凝血酶受体 (TR)基因的反义位置 .半定量RT PCR证实TR基因表达降低 ,p2 1基因表达升高 ,ATR和p2 1(AP)双基因得到了共表达 .以具有可提供复制和包装功能的重组单纯疱疹病毒rHSV rc/ΔU12分别感染载有不同基因的BHK细胞株 ,包装产生重组AAV (rAAV)病毒 ,并经点杂交法测定其滴度 (每毫升病毒液中所含病毒颗粒数 ) .rAAV/AP中ATR与 p2 1的病毒颗粒数分别为 1 0 2× 10 13 /ml和 1 0 8× 10 13 /ml,单基因rAAV/ATR的滴度为 6 5 4× 10 12 /ml,rAAV/P2 1为 1 0 6× 10 13 /ml,为进一步的体内外实验奠定了物质基础 .In order to study the co expression effect of antisense thrombin receptor (ATR) and p21 genes on preventing restenosis, an adeno associated virus(AAV) vector carrying the two genes with different promoters was constructed. Simultaneously, rAAV vectors containing ATR or/and p21 gene were constructed respectively. BHK 21 cell lines carried different plasmids were established by lipofectamine transfection and G418 selection. During the process of cell clones selected, double gene showed stronger inhibition to cell proliferation than single gene. The speed of clones formation decreased meanwhile the morphological alteration of cells occurred. Southern blot confirmed that expression vectors containing ATR or/and p21 gene had been steadily integrated into BHK 21 cells. The antisense position of TR gene has been maintained. Semi quantitative RT PCR showed that the expression of TR decreased and the expression of p21 increased. Co expression of double gene was obtained. rAAV virions were packaged through recombinant herpes simplex virus (rHSV rc/ΔU12) infecting BHK 21 cell lines carried various vectors. The titre of rAAV (particles/ml) determined by dot blot analysis. The particles of ATR and p21 in rAAV/AP double gene vector were 1 02×10 13 particles/ml and 1 08× 10 13 particles/ml respectively. The particles of single gene rAAV /ATR or rAAV /p21 were 6 54×10 12 particles/ml or 1 06×10 13 particles/ml.

关 键 词：反义凝血酶受体 p21双基因 共表达 腺病毒伴随病毒载体 构建 包装 再狭窄 

分 类 号：R543[医药卫生—心血管疾病] R394.8[医药卫生—内科学]