Construction of genetic recombinant adenovirus carrying human growth and differentiation factor-5 gene by using AdEasy-1 adenovirus vector system
10.3969/j.issn.1671-8348.2014.12.002
- VernacularTitle:应用AdEasy-1腺病毒载体系统构建人GDF-5基因重组腺病毒
- Author:
Xuwei LUO
;
Kang LIU
;
Zhu CHEN
;
Ming ZHAO
;
Xiaowei HAN
;
Yiguang BAI
;
Gang FENG
- Publication Type:Journal Article
- Keywords:
adenoviridae;
grow th and differentiation factor-5;
gene therapy
- From:
Chongqing Medicine
2014;(12):1412-1415
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the genetic recombinant adenovirus vector carrying the human growth and differentiation fac-tor-5(GDF-5) gene by using AdEasy-1 adenovirus vector system and to amplify and prepare the recombinant adenovirus in HEK 293 cells .Methods Human GDF-5 gene obtained by PCR was inserted into plasmid pMD19-T and the 1 .7 kb GDF-5 cDNA sequence was cloned into the adenoviral shuttle plasmid pShuttle-cytomegalovirus(CMV) of the AdEasy-1 system .It was identified by DNA sequencing and a digestion with Hind Ⅲ restriction enzyme .The resultant pShuttle-CMV-GDF-5 was used to generate the adenovi-ral GDF-5 vector through homologous recombination with the adenoviral backbone plasmid ,pAdEasy-1 in BJ5183 bacterial cells .It was selected by kanamycin and identified by a digestion with Hind Ⅲ restriction enzyme and amplified in XL10-Gold competent bac-teria .The DNA of recombinant adenovirus vector was finally linearized by Pac Ⅰ and the adenoviral recombinants were used to pro-duce adenoviruses in HEK293 packaging cells ,resulting in an Ad-GDF-5 identified by Western blot .The virus titer was assayed by TCID50 .Results GDF-5 cDNA sequence obtained by PCR was 1 .7 kb .Gene sequencing results indicated that the sequence was i-dentical with the one in GENBANK .Cloned sequence 1 .7 kb(GDF-5) was obtained by a digestion with Hind Ⅲ restriction enzyme after GDF-5 cDNA segment was cloned into pShuttle-CMV and AdEasy-1 .Western blot showed that two bands migrating at ap-proximately 15 and 55 kDa were clearly observed in PVDF membrane .These data confirmed that HEK293 cells expressed a large number of mature GDF-5 protein after infected with Ad-GDF-5 .Our research results demonstrated that recombinant adenovirus vector GDF-5 was successfully constructed .The virus titer was 5 .6 × 109 PFU/mL .Conclusion Recombinant adenovirus vector carrying the human GDF-5 gene is successfully constructed by using the AdEasy-1 adenovirus vector system .Moreover ,the Ad-GDF-5 with high titer is prepared .These provide the basis for further study of the biological function of GDF-5 and the gene thera-py of its related diseases .
