生长激素释放因子和垂体腺苷酸环化酶激活肽基因的共表达  

Co-expression of Growth Hormone Releasing Factor and Pituitary Adenylate Cyclase Genes

作　　者：刘松财[1] 任晓慧[1] 张明军[1] 戴建威[1] 郝琳林[1] 张永亮[1] 张玉静[1] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062

出　　处：《科技导报》2006年第8期36-39,共4页Science & Technology Review

基　　金：国家自然科学基金资助项目(30270973)

摘　　要：通过PCR扩增化学合成了PACAP和GRF基因。构建了PACAP、GRF单基因和PACAP-GRF双基因真核表达载体;通过脂质体介导3种真核表达载体在CHO细胞均有表达,RT-PCR、Dot-blot、Western-blot结果阳性,通过检测证明表达产物具有生物学活性;以PLGA为载体,采用复乳-液中蒸发法将3种重组质粒制备成微球,通过肌肉注射给小鼠和獭兔,实验结果表明实验组累积增重与生理盐水组比差异极显著(P<0.01),双基因组增重效果优于单基因组;双基因组对IGF-1促分泌作用也高于单基因组。首次证明了PACAP和GRF双基因共表达,对动物的促生长具有协同作用。PACAP coding gene was obtained with chemical synthesis and PCR amplification. The eukaryon expression vectors, pIRES1-PACAP, pIRES1-GRF and pIRES1-PACAP-GRF, were constructed respectively, then the three expression vectors were transfected by Lipofectin to prepared CHO cells. The total RNA was extracted from transfected CHO cells,and the positive result was obtained by RT-PCR, Dot-ELISA and Western blot. Rats were treated with 0.5 ml of transfection supernatant by muscle injection. After 8 hours, IGF- lconcerntration in P-G-P group was significantly higher than that of the other groups（P 〈 0.05）. Three expression plasmids were extracted and poly lactic-co-glycolic acid （PLGA） microspheres encapsulating plasmids were prepared by double emulsion-in hliquid evaporation process. Mice were muscularly injected with plasmid microspheres, bare plasmid and saline. After 20 days, the mean accumulative body weight gain in P-G-P microsperes group was the highest, increased by 64.71%（P〈0.01 ）, 28.78%（P〈0.01 ）, 32.72%（P〈0.01 ）, and 20.78%（P〈0.05）to groups injected with saline, P-G microspheres, P-P microspheres and bare plasmids, respectively. Rabbits were treated with three plasmid microspheres （1 mg plasmid/kg of BW）and saline by muscular injection. After 30 days, the mean accumulative body weight gain in plasmid microspheres groups were significantly higher than that of the group injected with saline. Among groups, the accumulative body weight gain in P-G-P group was the highest,increased by 81%（P〈0.01 ）, 15%（P〈0.05）and 7%（P〉0.05） to the saline group, P-G microspheres group and P-P microspheres group, respectively. The concentrations of serum IGF-1 increased in all plasmid microspheres injection groups, which were significantly higher than that of the saline injection group. We draw a conclusion that GRF and PACAP co-expression in animal body do increase IGF-1 concentration and improve animal growth,whose biological activity might be more powerful than GRF and PACAP si

关 键 词：GRF PACAP 基因转染 微球 

分 类 号：S852.2[农业科学—基础兽医学]