CH50真核表达载体pCH503的构建及小鼠体内表达的趋化与抗肿瘤活性

构建重组 FN多肽 CH50真核表达载体并在小鼠体内表达 ,研究其趋化与抗肿瘤作用 .采用重组 DNA技术构建表达质粒 ;体内进行基因转染 ,采用 RT- PCR鉴定导入基因的表达 ;通过肝素亲和层析、SDS- PAGE和 Western blot鉴定表达产物 ;腹腔细胞计数、Giemsa染色分析以及肌肉组织切片与染色观察体内基因转染后的趋化作用 ;小鼠黑色素瘤模型研究基因转染抑制肿瘤的作用 .从 CH50原核表达载体获得重组多肽的 c DNA,5′端加上小鼠 IFN- 5′端非编码区和信号肽编码区的 c DNA,3′端加上人 FN c DNA的 3′端非编码区 ;将重组 c DNA插入 p REP8质粒 ,即构建出p CH50 3质粒 .巨噬细胞在体内经 p CH50 3转染 ,然后在体外培养 ,能够产生 CH50多肽 .以p CH50 3分别进行腹腔基因转染和肌肉内基因转染 ,均可对免疫细胞产生趋化作用 ;p CH50 3体内转染可以使小鼠腹腔内黑色素肿瘤结节数降低 50 %～ 60 % . CH50真核表达载体 p CH50 3可在小鼠体内表达 ,体内基因转染可趋化免疫细胞和抑制肿瘤结节形成 ,在肿瘤综合治疗中有重要意义 .

Construction of Eukaryotic Expressing Vector pCH503 of CH50 and Its Chemotaxis and Anti-tumor Function by Expression in vivo in Mice

An eukaryotic expressing vector which expresses CH50,a recombinant polypeptide of human fibronectin,in mice was constructed,and its chemotaxis and anti tumor function after in vivo gene transfection were investigated.The plasmid was constructed by recombination techniques.Gene transfection was performed in vivo .The expression of the transfected gene was identified by RT PCR.The expressed product was identified by heparine affinity chromato graphy,SDS PAGE and Western blot.The chemotaxis after transfection with pCH503 in vivo was observed by counting and Giemsa staining of coeliac cells and histotomy and staining of muscle tissue.The inhibition of gene transfection of pCH503 on molenoma was observed in mice.The cDNA fragment coding CH50 polypeptide from a prokaryotic expressing vector of CH50 was ligated with 5′ terminal noncoding region and signal peptide coding region of mouse IFN  cDNA at 5′ side and 3′ terminal noncoden region of human FN cDNA at 3′ side.The recombinant cDNA was inserted into plasmid pREP8.The resulting expressing plasmid was designated as pCH503.The macrophages transfected with pCH503 in vivo and cultured in vitro could produce CH50.The chemotaxis on immune cells was observed after transfection of pCH503 either in peritoneal cavity or in muscle.The number of melanoma nodes in mice was reduced by 50%-60% after coeliac transfection with pCH503.The pCH503,a eukaryotic expressing vector of CH50,could express in vivo in mice.The transfection of pCH503 in vivo had the chemotaxis on immune cells and could inhibit the formation of tumor nodes,suggesting that plasmid pCH503 is potentially useful in combined treatment of tumor.