Construction of CD44 gene overexpression lentiviral vector and its expression in LNCaP cells / 中华老年医学杂志

ABSTRACT

Objective To construct an overexpression lentiviral vector of CD44 and obtain an androgen-dependent prostate cancer LNCaP cell line with CD44 overexpression.Methods CD44 gene sequence was obtained by PCR amplification.The synthesized oligonucleotides were cloned in the lentiviral vector LV5 after digested with NotI and NsiI enzyme.Recombinant plasmids were verified by enzyme digestion analysis and sequencing,and were transfected into 293T package cells.Supernatant was collected and concentrated to obtain lentivirus solution at a high titer.The titer of virus was identified by multiple proportion dilution methods.Then the recombinant viruses were transfected into LNCaP cells.Results Fluorescence intensity in infected LNCaP cells was increased,detected by a fluorescence microscope.And QRT PCR and Western blot results showed that the expression of CD44 was significantly increased(P＜0.05).Conclusions The overexpression lentiviral vector of CD44 is successfully constructed,and the LNCaP cell line with CD44 overexpression is obtained.