Cloning of human uroplakin II gene from Chinese transitional cell carcinoma of bladder and construction of its eukaryotic expression vector.
To clone Uroplakin Ii gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GIII / T3N0M0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin II gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between Xba I and Hind III restrictive sites of eukaryotic expression vector pcDNA3. 0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin II negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin II were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin II cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100% homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UP II for Uroplakin II was successfully constructed. After being transferred with pcDNA-UP II for 72 h, cellular Uroplakin II mRNA levels were significantly improved (P < 0.01). It is concluded that human Uroplakin II gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin II gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.
Chen
Fangmin
F
Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Zeng
Fuqing
F
Tong
Qiangsong
Q
Zheng
Liduan
L
Wang
Liang
L
Dong
Jihua
J
Lu
Gongcheng
G
D001483:Base Sequence; D002295:Carcinoma, Transitional Cell / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008875:Middle Aged; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D001749:Urinary Bladder Neoplasms / Q000235:genetics*; D060170:Uroplakin II
D018076:DNA, Complementary; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C554658:UPK2 protein, human; D060170:Uroplakin II