CAJ | 학술논문

目的：应用AdEasy-1腺病毒载体系统构建人生长分化因子-5（GDF-5）基因重组腺病毒载体，并在 HEK293细胞中扩增制备重组腺病毒。方法将PCR获取的GDF-5基因插入到质粒pMD19-T中，基因测序鉴定后将目的基因克隆至腺病毒穿梭质粒pShuttle-CM V ，Hind Ⅲ酶切鉴定。重组质粒经 PmeⅠ酶切使之线性化并去磷酸化处理，电转化含腺病毒骨架质粒pAdEasy-1的感受态大肠埃希菌BJ5183，使其在细菌内发生同源重组，卡那霉素筛选，Hind Ⅲ酶切鉴定阳性克隆，扩增重组腺病毒质粒，并转染 HEK293细胞，包装成重组腺病毒Ad-GDF-5，分别采用Western blot、TCID50法对Ad-GDF-5进行蛋白鉴定和滴度测定。结果经PCR扩增得到的GDF-5大小约为1．7 kb ，基因测序结果证实该基因与GENBANK中人GDF-5基因序列相同。pShuttle-CM V-GDF-5、pShuttle-CM V-GDF-5-AdEasy-1经 Hind Ⅲ酶切后均得到大小约为1．7 kb的片段，与GDF-5大小相符。Western blot证实Ad-GDF-5感染HEK293细胞后大量表达成熟GDF-5蛋白。研究结果表明成功地构建了携带人GDF-5基因的重组腺病毒载体，并制备出5．6×109 PFU/mL的重组腺病毒。结论利用AdEasy-1腺病毒载体系统成功构建了携带人GDF-5基因的重组腺病毒载体，并制备出高滴度值的重组腺病毒Ad-GDF-5，为进一步研究GDF-5的生物学功能及其相关疾病的基因治疗奠定了基础。
목적：응용AdEasy-1선병독재체계통구건인생장분화인자-5（GDF-5）기인중조선병독재체，병재 HEK293세포중확증제비중조선병독。방법장PCR획취적GDF-5기인삽입도질립pMD19-T중，기인측서감정후장목적기인극륭지선병독천사질립pShuttle-CM V ，Hind Ⅲ매절감정。중조질립경 PmeⅠ매절사지선성화병거린산화처리，전전화함선병독골가질립pAdEasy-1적감수태대장애희균BJ5183，사기재세균내발생동원중조，잡나매소사선，Hind Ⅲ매절감정양성극륭，확증중조선병독질립，병전염 HEK293세포，포장성중조선병독Ad-GDF-5，분별채용Western blot、TCID50법대Ad-GDF-5진행단백감정화적도측정。결과경PCR확증득도적GDF-5대소약위1．7 kb ，기인측서결과증실해기인여GENBANK중인GDF-5기인서렬상동。pShuttle-CM V-GDF-5、pShuttle-CM V-GDF-5-AdEasy-1경 Hind Ⅲ매절후균득도대소약위1．7 kb적편단，여GDF-5대소상부。Western blot증실Ad-GDF-5감염HEK293세포후대량표체성숙GDF-5단백。연구결과표명성공지구건료휴대인GDF-5기인적중조선병독재체，병제비출5．6×109 PFU/mL적중조선병독。결론이용AdEasy-1선병독재체계통성공구건료휴대인GDF-5기인적중조선병독재체，병제비출고적도치적중조선병독Ad-GDF-5，위진일보연구GDF-5적생물학공능급기상관질병적기인치료전정료기출。
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 .