Cloud-Clone Corp.develops the recombinant protein,antibody and assay kit for CUZD1 research

CUZD1 is strictly regulated by estrogen in two key reproductive tissues, the uterus and oviduct. Spatio-temporal analyses reveal that CUZD1 mRNA is expressed in a highly stage-specific manner in the uterus and oviduct, and its expression is restricted to the surface epithelium of both of these tissues. Nucleotide sequence analysis of the full-length CUZD1 cDNA indicates that it has an open reading frame of 1821 nucleotides encoding a putative protein of 607 amino acids with a single transmembrane domain and a short cytoplasmic tail. Although the precise function of CUZD1 is not clear, presently, its unique pattern of expression in the uterus and oviduct, and its regulation by estrogen, a principal reproductive hormone, lead us to speculate that this novel gene plays an important role in events during the reproductive cycle and early pregnancy. CUZD1 antiserum inhibits cell attachment and proliferation of ovarian cancer cells, so the protein may be involved in these processes. And CUZD1 may also play a role in the uterus during late pregnancy and/or in trypsin activation in pancreatic acinar cells.

There is few commercial CUZD1 products on the market, for this important protein, Cloud-Clone Crop. produced a series of products, containing protein, antibody and ELISA kit for the researchers who are interested in this research area.

After analyzing of antigen epitope prediction, function/structure domain, mRNA secondary structure prediction, recombinant protein soluble forecast and other so on, Pro218 ~ Arg479 of human CUZD1 were chose as the fragment for expression by E.coli., the fragment belong to the extracellular domain, with strong immunogenicity, and it gives soluble expression in Escherichia coli. Upstream and downstream primers were designed according to the nucleic acid sequence of Q86UP6 in http://www.uniprot.org/. Two Restriction enzyme sites were designed at the 5’ of the two primer for recombinant vector construction.

[ PRIMER INFORMATION ]
Upstream Primer: CGgaattcCCCTCCACCAACTCTGGC
Downstream Primer: CCGctcgagCTATCTCCCATAGTGTCCAAATAAGG

High fidelity enzyme was used for PCR to obtain a 800 bp cDNA fragments. The cDNA fragment was sequenced and recombinant with prokaryotic expression vector pCC-GST by double enzymes restriction and ligation. Then the recombinant vector was transform in to BL21 (DE3) for protein expression. The sequencing results are shown in figure 1.

The vector pCC - GST is a prokaryotic recombinant expression vector, which was produced by our company independently. There are two fusion expression tags in the vector, his tag and GST tag, so, the recombinant proteins can be purified by two different methods, and the two tags in the vector could also improve the solubility and immunogenicity of the recombinant proteins. There is a protease enzyme site between expression tags and the MCS, the enzyme site can be used for removing of the expression tags.

After IPTG induced, we got a recombinant protein with 59 kda MW (Cat.No.RPQ635Hu01), the result were show in figure 2.and the corresponding protein sequence was show in figure 3.

Based on BLASTp in the NCBI website, the similarity of the fragment we got and human CUZD1 (NP_071317.2.) is 100%., which means we get the correct recombinant protein fragment, shown in figure 4, as follows:

The recombinant human protein CUZD1 was purified by Nickel column. The monoclonal antibody of human CUZD1 was produced by mice, and the polyclonal antibody was produced by rabbit. The two different antibodies were purified by affinity chromatography and the product Cat. no. are MAQ635Hu21 and PAQ635Hu01.

We checked the targeted signal in human primary pancreatic adenocarcinoma cell line of BXPC-3, but the non-specific bands also existed. We got the target bands of the expected size in the pancreatic tissue of mice and rats, no signal detected in rats ovary. And the obvious signal can be observed in the human pancreas cancer tissue by IHC detection. The experiments result show that we get high-quality human CUZD1 antibodies.

The ELISA Kit for CUB And Zona Pellucida Like Domains Protein 1 (CUZD1)，SEQ635Hu were also produced by the recombinant protein and antibodies. There are two antibodies used in kit. The capture antibody used in the kit is monoclonal antibody, and the detection antibody is polyclonal antibody. the standard of kit is the recombinant human CUZD1 (Pro218~Arg479) without expression tag. The sensitivity and specificity of the kit is very high, and the kit was suitable for CUZD1 detection in human samples.The fig 7 is the detection range and sensitivity of CUZD1 Assay kits. And the fig 8 is the typical standard curve of it.