金鱼生长激素Ⅱ基因在杆状病毒中的表达

Gene Expression of Gold Fish Growth Hormone Ⅱ in Baculovirus

以ｐＳＸＩＶＶＩ＋Ｘ３为转移载体，将编码金鱼生长激素Ⅱ的ｃＤＮＡ插入粉纹夜蛾核型多角体病毒（ＴｎＮＰＶ）基因组中，构建了重组病毒株ＴｎＮＰＶＳＸ＋ｇｆＧＨⅡ４６。该毒株能在草地贪夜蛾（Ｓｐｏｄｏｐｔｅｒａｆｒｕｇｉｐｅｒｄａ）离体培养细胞及银纹夜蛾（Ａｇｙｒｏｇｒａｍｍａａｇｎａｔａ）幼虫中表达金鱼生长激素基因。蛋白免疫印迹表明，表达的生长激素蛋白分子量为２２．５ｋＤａ，与理论计算值相符，且表达的生长激素可分泌到感染细胞的培养基及虫体血淋巴中。ＲＩＡ结果表明，表达产物与天然的生长激素有相似的免疫特性，重组病毒在感染细胞９６ｈｐｉ所表达的生长激素达到最高水平，平均每１０５个细胞可在细胞培养基中检测到金鱼生长激素Ⅱ达８６．７４ｎｇ；平均每克干虫可产生金鱼生长激素Ⅱ２１４μｇ。

Using a transfer vector plasmid pSXIVVI +X3 without an initiation codon, the occluded recombinant Trichoplusia ni nuclear polyhedrosis virus as an expressing vector carrying the cDNA encoding gold fish growth hormone Ⅱ(gf GHⅡ) under the control of the SynXIV promoter has been constructed. Immunoblot analysis revealed that the virus mediated gfGH Ⅱ can be detected as early as 24 hr pi and the expression level reached to the highest in 96 hr pi in the Sf cells and culture medium or larvae and haemolymph, the molecular weight of the expressed protein is 22.5 kDa, which is equivalent to the value calculated from the predicted amino acid sequence. The expression level in vivo and in vitro was quantified using RIA. Average 10 5 Sf9 cells may secret gfGH Ⅱ into medium reaching level of 86.74 ng. The expression level of gfGHⅡ in larvae may reach to 214μg per gram of dry larvae.