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[Cloning of tight junction protein claudin-1 and construction of the mammalian expression vector]
Lin Chen1, Bo Jiang, Ya-li Zhang
1Institute for Digestive Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. clin66666@yahoo.com.cn
Objective:
To construct a recombinant plasmid containing the coding region of tight junction protein claudin-1 gene to understand the functional role of claudin-1 in human colorectal carcinoma.
Methods:
The total RNA was extracted using Trizol from human colorectal carcinoma cell line SW620, and the DNA for claudin-1 was obtained by means of RT-PCR. The PCR product was inserted into the plasmid pEGFP-C1 after restriction endonuclease digestion and ligation. The recombinant plasmid was then transfected into human colorectal carcinoma cell line SW480.
Results:
The sequence of the recombinant plasmid was verified by restriction endonuclease and DNA sequence analysis, and the target protein expression was detected mostly on the cell membrane.
Conclusion:
The expression vector claudin-1/pEGFP-C1 has been constructed successfully and the target protein can be expressed in human colorectal carcinoma cell line.
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