P1/Fas-CCL19双表达重组腺病毒载体的构建及表达鉴定

 目的
利用pAdEasy-1 腺病毒包装系统构建血型B抗原模拟多肽P1/Fas-CCL19双表达重组腺病毒，并在体内外试验中验证其表达及可能
的疗效。方法应用腺病毒穿梭质粒pShuttle-CMV、骨架质粒pAdEasy-1构建重组腺病毒质粒pAd-CMV-P1/Fas-CCL19并转染至
HEK293A细胞，获得重组腺病毒Ad-P1/Fas-CCL19颗粒后进一步感染乳腺癌4T1细胞并鉴定转染4T1细胞内P1/Fas-CCL19 mRNA和蛋
白质的表达。构建经人B型血红细胞免疫的4T1荷瘤小鼠模型，比较瘤内注射重组腺病毒Ad-P1/Fas-CCL19组、空腺病毒组、0.9%
氯化钠溶液组的荷瘤小鼠的肿瘤变化及生存期。结果感染后4T1 细胞中P1/Fas-CCL19 mRNA和蛋白质均有表达。瘤内注射重组腺
病毒Ad-P1/Fas-CCL19较瘤内注射空腺病毒、0.9%氯化钠溶液明显抑制4T1肿瘤的生长，但三组小鼠的生存期无明显区别。结论
P1/Fas-CCL19重组腺病毒载体成功构建，体内外实验均证明了其稳定表达及有效性，为进一步临床研究奠定了基础。

Construction and Identification of PI/Fas-CCL19 Double Expressed Reorganization Adenovirus Carrier

Objective
To construct recombinant adenovirus expressing both simulated peptide of blood group B antigen P1/Fas and CCL19
by pAdEasy-1 packaging system,and test their expression.MethodsWe constructed pShuttle-CMV-P1/Fas-CCL19 by
adenovirus Shuttle plasmid pShuttle-CMV and pAdEasy-1.Then，we transfected pAd-CMV-P1/Fas-CCL19 to HEK293A cells
and obtained recombinant Ad-P1/Fas-CCL19 particles which can infect 4T1 cells.At last we detected mRNA and
protein expression of P1/Fas-CCL19 in 4T1 cells.4T1 tumor-bearing mice model immuned by the B red blood cell was
established successfully,and injected with recombinant adenovirus Ad-P1/Fas-CCL19,empty adenovirus,saline into
the tumors,respectively.Then,tumor changes and survival period of the three groups were compared.ResultsP1/Fas-
CCL19 can be effectively expressed in 4T1 cells on both mRNA and protein levels.Intratumor injection of
recombinant adenovirus Ad-P1/Fas-CCL19 group inhibited 4T1 tumor growth significantly compared with intratumoral
injection of adenovirus,empty saline,but survival time has no obvious difference among the three
groups.ConclusionP1/Fas-CCL19 recombinant adenovirus vector was successfully constructed,experiments proved its
stable expression and efficacy in vivo,in vitro，which make a foundation for further clinical research.