Objective: To make sure that 5D4 cDNA is a full length cDNA and to prepare antigens to make monoclonal antibodies against different epitopes in 5D4 protein.
Methods: Recombinant expression vectors pGEX-5X-3-5D4 (full length) and pGEX-5X-3-5D4 (5'fragment) were constructed. 5D4 polypeptides were expressed in Escherichia coli DH5 alpha. 5D4 mRNA expression was detected in various tissues and cell lines with Northern blot assay and RT-PCR.
Results: Northern blot demonstrated five mRNA transcripts of 1.9, 2.4, 3.9, 4.5, and 10.0 kb in length existing and expressing of various extent in various tissues. The 1.9 kb mRNA transcript was observed only in the spleen and the small intestinal tissues. The expression of 5D4 mRNA was different in various cell lines. 5D4 polypeptides were expressed in Escherichia coli DH5 alpha.
Conclusion: The 1,846 bp 5D4 cDNA is a full length cDNA. A full length 5D4 protein and a 5D4 polypeptide which is the NH2 side to the transmembrane region fused with GST with a molecular weight of 66,000 and 60,000 expressing in Escherichia coli DH5 alpha.