Construction Of Co-Expression Plasmid PBbudCE4.1-CD44-ALDH1and Its Expression In Vitro

Objectives:This experiment is to construct the mammalian CO-expression plasmid pBudCE4.1-CD44-ALDH1, plasmid pBudCE4.1-CD44and plasmid pBudCE4.1-ALDH1, which were transfected into MCF-7respectively. The expression of CD44and ALDH1were detected in each group so as to compare double gene group with single gene group respectively. Theoritcal supports were provided to CD44and ALDH1-based targeted cancer therapy research by this experiment.Methods:1Culture three tumor cell lines of MCF-7, PC3, A542, then detect the expression of the gene CD44and ALDH1by RT-PCR.2Based on the results of RT-PCR test, Fragments of CD44antigen isoform4precursor gene with NotI/Xhol restriction sites and Fragments of ALDH1gene with Hindlll/Xbal restriction sites were gained from MCF-7and A542.3The mammalian CO-expression plasmid pBudCE4.1-CD44-ALDH1, plasmid pBudCE4.1-CD44and plasmid pBudCE4.1-ALDH1were constructed by Recombinant DNA technology.4The recombinant plasmids were identified by the methods of PCR, restriction endonuclease digestion and DNA sequence analysis.5MCF-7cell line was transfected with this Co-expression plasmid using lipofectin reagent according to the transfected situation, the MCF-7cells were divided into5groups, the non-transfected group(Group A), the group transfected by empty vector(Group B), the group transfected by CD44(Group C), the group transfected by ALDH1(Group D)and the group transfected both CD44and ALDH1(Group E).The expression of CD44and ALDH1were detected by RT-PCR and Western blot technique, the expression differences were analysed at the end. 6The effects of CD44and ALDH1on the proliferation of MCF-7cells were detected by CCK-8assays. With MTT assays, the adhesive ability to matrigel and FN was measured.Results:1The result of RT-PCR showed:the expression of CD44gene in MCF-7and PC3cells, no in A542cells; the expression of ALDH1gene in A542cells, no in MCF-7and PC3cells.2The gene of CD44antigen isoform4precursor and ALDH1were constructed by RT-PCR technology.3The mammalian CO-expression plasmid pBudCE4.1-CD44-ALDH1, plasmid pBudCE4.1-CD44and plasmid pBudCE4.1-ALDH1were obtained successfully.4The recombinant plasmids were identified by the methods of PCR, restriction endonuclease digestion and DNA sequence analysis successfully.5The results of RT-PCR and Western blot were measured with the grey value. To the expression of protein of CD44, the differences between groups A、B and groups D、E were significant (P<0.05).The differences between groups C and E were significant (P<0.05); To the expression of protein of ALDH1, the differences between groups A、B and groups C、D、E were significant (P<0.05).the differences between groups D and E were significant (P<0.05).6The recombinant plasmids were transiently transfected into MCF-7cells. As compared with groups A、B, groups C、D revealed a marked raise in tumor proliferation and adhesive ability (P<0.05).And the effects with groups E were higher than groups C、D (P<0.05).Conclusion:1The mammalian CO-expression plasmid pBudCE4.1-CD44-ALDH1, plasmid pBudCE4.1-CD44and plasmid pBudCE4.1-ALDH1were obtained successfully.2The results indicated that CD44and ALDH1gene and their expression products play significant roles in promoting proliferation and adhesive ability of MCF-7cells in vitro. 3There are good prospects that the recombinant plasmid pBudCE4.1-CD44, pBudCE4.1-ALDH1, pBudCE4.1-CD44-ALDH1would be used in a variety of tumor basic researches.