摘要
目的 :构建并鉴定细胞因子mGM CSF和肿瘤特异抗原P1A共表达载体 ,为研究新型的肿瘤疫苗提供新的策略。方法 :以PCR方法从pCI neo P1A中扩增出P1A编码基因片段 ,构建于pRSC质粒上 ;再从pORF mGM CSF中扩增出mGM CSF基因片段 ,插入pRSC P1A重组质粒P1A基因的上游。以酶切和DNA测序进行鉴定。并将鉴定过的重组质粒以脂质体法转染 772 1细胞 ,用RT PCR法鉴定转染细胞中P1A基因和mGM CSF基因的表达。结果 :经酶切鉴定和DNA测序证实mGM CSF和P1A重组质粒构建正确 ,并在转染此质粒的 772 1细胞中检测出了P1A和mGM CSF基因的表达。结论 :成功构建mGM CSF和P1A的共表达载体 。
Objective To construct the coexpressing vector of pRSC-mGM-CSF/P1A containing genes of P1A and mGM-CSF for the purpose of developing tumor vaccine.Methods P1A gene was amplified from pCI-neo/P1A vector by PCR and was ligated into vector pRSC to form pRSC-P1A recombinant.The mGM-CSF gene which was obtained from pORF-mGM-CSF vector by PCR was inserted to the upstream of the P1A in pRSC-P1A recombinant to develop a pRSC-mGM-CSF/P1A tumor vaccine constructs.After the identified recombinant plasmids were transformed into cell 7721 with lipofectamine,the expression of P1A and mGM-CSF in the cells were determined with RT-PCR. Results The pRSC-mGM-CSF/P1A tumor vaccine constructs was identified by restrictive digestion and sequencing methods respectively, and the expression of P1A gene and mGM-CSF gene could be detected in the transformed 7721 cells.Conclusion The recombinant pRSC-mGM-CSF/P1A has been successfully constructed,and it provides a good foundation for developing novel tumor vaccine.
出处
《东南大学学报（医学版）》
CAS
2004年第3期184-188,共5页
Journal of Southeast University(Medical Science Edition)
