用于骨移植的hCbfa1重组腺病毒载体的构建

Construction of hCbfa1 Recombinant Adenovirus Vector for Bone Transplant

克隆hCbfa1的cDNA基因,构建其重组腺病毒载体,以用于研究hCbfa1腺病毒载体在人造骨移植中的作用.将hCbfa1的cDNA目的片段插入腺病毒柯氏质粒PAxCAwt中,再将此质粒与腺病毒DNA末端蛋白复合物共转染293个细胞,通过同源重组构建hCbfa1重组腺病毒载体.重组得到的阳性克隆经酶切、测序鉴定正确,包装纯化后,检测得病毒滴度为2×109PFU/mL,从而成功地构建了hCbfa1重组腺病毒载体,为下一步应用到人造骨移植的转基因治疗打下了基础.

In order to investigate the effect of hCbfal adenovirus vector on the artificial bone transplant, hCbfal cDNA gene was cloned to construct the recombinant adenovirus vector. In the construction, the target fragment of hCbfal cDNA was first inserted into cosmid PaxCAwt and then cotransferred into 293 cells with adenovirus gene DNA TPC （Terminal Protein Complex） by means of homologous recombination to construct the recombinant adenovirus vector. The positive clones of the recombinants were screened by enzyme digest identification and were then treated by sequencing and purifying. A concentration of viral liquids of 2 × 10^9 PFU/mL was thus detected, which led to the successful construction of hCbfal recombinant adenovirus vector. The present study lays a foundation for the further application of the vector to the transgenic therapy for artificial bone transplant.