用构建的新型BmNPV载体在家蚕高效表达人β-干扰素

High Level Expression of the Human Beta-interferon Gene in the Silkworm with New Constructed BmNPV Vector

构建了家蚕核多角体病毒新型载体ｐＢｍ９２，该载体将多角体蛋白基因的起始密码ＡＴＧ改变为ＡＴＴ，然后在多角体蛋白基因的＋１２位后连接有５个外源基因的克隆位点。将ＨｕＩＦＮ－β基因克隆在多角体蛋白基因的＋１２位后，构建了ｐＢｍＩＦＮ＋１２；同时构建了Ｈｕ ＩＦＮ－β克隆在－３位后的转移载体ｐＢ－ｍＩＦＮ－３。

The Bombyx mori nuclear polyhedrosis virus (BmNPV) and Bombyx mori cells as well as silkworm larvae were used successfully for production of biologically active recombinant proteins. But the types of BmNPV general vectors are fewer. There-fore, new type vector plasmid pBm92 was constructed in this experiment. The transla-tional initiation codon ATG of the polyhedrin gene in pBm92 was changed into ATT, then five cloning sites of foreigen gene ligated after + 12 bp of polyhedrin gene. Human beta-interferon(HuIFN-β)gene was cloned into.pBm92,constructing pBmIFN + 12;mean while we constructed the transfer vactor pBmIFN-3 in which HuIFN-B gene was cloned after-3 bp of the polyhedrin gene. Two types of transfer vector DNAs and BmNPV gemonic DNA were cotransfected Bm-N cells respectively. Recombinand viruses were screened with the character of recombinant viruses no producing polyhedrin in the virus plaque assay, and identified by the hybridization of recombinant virus DNA with HuIFN-β gene probe. IFN activity of media of Bm-N cells infected by recombinant virus BmIFN + 12 was 2. 0× 106IU/ml,and IFN activity of haem olymph of silkworm larvae infectd by BmIFN + 12 was 5.0× 107IU/ml. Expression level of BmIFN + 12 was more than 2 - 4 times that of BmIFN - 3. rHuIFN-β produced by Bm-N cells and silkworm larvae has the antigenity identical to the native HuIFN-β.