Experimental Study on Construction of Human Hiwi Adenovirus Vector and Apoptosis of K562 Cells Induced by Transfected Vector

Leukemia stem cells are responsible for the genesis, progression, drug resistance and relapse of leukemia. Hiwi gene is an important divisive regulatory factor, which plays a role in maintaining the resting stage and down-regulating the cell cycle of stem/progenitor cells. We acquired human overall Hiwi coding region genes and constructed an adenovirus vector carrying human Hiwi with fluorescin. This study will not only establish the foundation of the further study in function and mechanism of Hiwi induce the differentiation and apoptosis of leukemia stem cells, but also provide the theoretical basis for searching new therapeutic target and method for leukemia.

Using the overlap extension PCR method to amplify overall Hiwi coding region genes and insert overall Hiwi coding region genes into Flag-IRES –hrGFP vector that carrying green fluorescin with Gateway clone technology to construct pDown-Hiwi-3×flag-IRES-hrGFP. After transform the vector to Stb13, we screen the positive clone with PCR method, extract the plasmid and process recombination reaction between pDown-Hiwi-3×flag-IRES-hrGFP clone vector and pAV.Des1d expression vector to abtain the pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP adenovirus vector recombinant. Screening the positive clone with PCR and extracting the plasmid which were digested by enzyme and then examined by sequencing consequences. At last packaged Ad-Hiwi-3×flag-IRES-hrGFP adenovirus vector recombinant is obtained. Transfect the recombinant adenovirus vector to K562 cells, and then identify the function of the cells.

Successfully clone human overall Hiwi coding region genes with the technology of overlap extension PCR and construct the adenovirus vector recombinant with enzyme cutting identification and gene sequencing examination.

Successfully construct Ad-Hiwi-3×flag-IRES-hrGFP adenovirus vector recombinant and establish the foundation for the further study of Hiwi gene in leukemia stem cells. The transfected recombinant adenovirus vector can induce K562 cells to apoptosis. [U1]ɾ³ý£z

No relevant conflicts of interest to declare.