Preparation of KIAA0100 Monoclonal Antibody

KIAA0100 introduction

Human KIAA0100 gene was identified from a cDNA library of human immature myeloid cell line KG-1 in 1995. It belongs to the HUGE database, which contains more than 2,400 members identified in the Kazusa cDNA sequencing project. Northern blot analysis showed its mRNA was expressed in the brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, thymus, spleen, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, HeLa cells, and KG-1 cells. In a study on breast cancer, breast cancer-overexpressed gene 1 (BCOX1) is an alternative splicing variant of human KIAA0100 gene, which encodes a putative protein of 222 amino acid residues with a calculated molecular weight of 24.920 kDa. Its mRNA expression was moderately elevated in ductal in situ carcinoma (DCIS), peaked in invasive breast carcinoma (IBC) and metastatic breast carcinoma cells (MET) whereas absent in benign ductal epithelial cells. Besides, high levels of BCOX1 expression were associated with poor prognosis in patients with invasive ductal carcinomas of the breast. Furthermore, microRNA-195 could suppress tumor cell proliferation and metastasis by directly targeting BCOX1 in prostate carcinoma, it can be seen that the KIAA0100 gene is closely related to breast cancer. However, human KIAA0100 protein's function and structure have remained unknown so far, and there is no confirmation of the molecular weight of the encoded protein has not been reported. Therefore, the preparation of efficient and specific KIAA0100 antibodies is an important tool to study the function and structure of KIAA0100 protein.

Fig 1. MAb recognized human KIAA0100 protein using immunocytochemistry analysis.
(Source: Journal of Pharmaceutical Analysis, July 2017.)

Preparation of KIAA0100 antibody

In another study, bioinformatics software was used to optimize Escherichia coli (E. coli) rare codons into E. coli normal codons of human KIAA0100 protein segment (1557～2234)，and then the synthetic optimal nucleotide sequence were cloned into prokaryotic expression vector pET-30a to construct the recombinant plasmid. After being identified by restriction enzyme digestion analysis，the recombinant plasmid were transformed into E.coli BL21( DE3) cells, and then the recombinant human KIAA0100 protein segment( 1557～2234) were expressed as a fusion protein with C-terminal 6 × histidine( His)-tag. The New Zealand white rabbits were next immunized by the recombinant protein, and successfully prepared an anti-human KIAA0100 antibody, and KIAA0100 antibody was used as primary antibody for protein blotting analysis, and the molecular weight of human KIAA0100 protein was confirmed to be 254 kDa. On the one hand, these results further demonstrated the antigenicity of the human KIAA0100 protein fragment (1557-2234), and on the other hand, they laid a solid foundation for further studies on the structure and function of human KIAA0100 protein.

Antibodies are the most versatile protein molecules in biology and medicine, and are central to the recognition of tumor antigens and the clarification of their structure and function. In the face of the unknown nature of the KIAA0100 gene, the preparation of KIAA0100 antibodies seems imperative to clarify whether it can be expected to be used as a therapeutic target in immunotherapy of tumors.

KIAA0100 Antibody