抗人CD28单链抗体基因的构建及在BmN细胞和家蚕中的表达

Construction of Anti-CD28 Single Chain Antibody Genes and Expression of the ScFv in BmN Cells and the Larvae of Bombyx mori

采用TP-PCR法,将抗人CD28单链抗体的重、轻链可变区基因和人工设计的昆虫杆状病毒多角体蛋白基因(ph)偏好的连接肽序列,拼接成抗人CD28单链抗体(抗CD28-ScFv)基因,并将其插入昆虫杆状病毒转移载体pBacPAK8的ph启动子,构建成重组转移载体pBacPAK8/CD28-ScFv。为便于表达产物的纯化,在CD28-ScFv的C端增加了6×Histag序列。通过脂质体介导法将重组转移载体pBacPAK8/CD28-ScFv和线性化病毒Bm-BacPAK6共转染BmN细胞,经空斑分析和蓝白斑筛选,获得重组杆状病毒Bm-BacPAK6 CD28-ScFv。将重组病毒Bm-BacPAK6 CD28-ScFv感染BmN细胞和5龄家蚕,SDS-PAGE和Western Blotting分析表明,在分子量约为28kD处有一表达带。BmN细胞的表达始于24h,表达峰在72h,96h时表达量开始下降,而5龄家蚕的表达始于48h,表达峰在120h。结果证明,抗人CD28单链抗体基因在BmN细胞和家蚕中得到了特异性表达。上述研究成果为抗人CD28基因工程抗体的研制奠定了基础。

The VH and VL gene fragments of anti-CD28 mAb were combined to form anti-CD28 ScFv gene by using TP-PCR method. Sequence analysis showed that 6 × His tag was added to it for the ease of purification and the VH and VL gene fragments were connected by a linker containing 15 amino acids which are biased by the baculovirus promoter, ph. Then ScFv gene fragment was inserted into baculovirus transfer vector pBacPAK8. The recombinant transfer vector, pBacPAK8/CD28-ScFv was constructed successfully. The pBacPAK8/CD28-ScFv and the linear Bm-BacPAK6 were co-transfected into the cell line of Bombyx mori （BmN） with the help of Lipofectin,then the product was purified by plaque assay and identified by PCR method. The recombinant virus, Bm-BacPAK6 CD28-ScFv, was obtained successfully. The BmN cells and the larvae of Bombyx mori were infected by the recombinant baculovirus and harvested every 24h postinfection. SDS-PAGE and Western Blotting analysis confirmed the expression of ScFv with the molecular weight of about 28kD. The expression in BmN cells was detected 24h post infection and it peaked at 72h, while in the larvae of Bombyx mori, the expression was detected 48h post infection and it peaked at 120h.