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Identification of binding epitope of a monoclonal antibody (Z12) against human TNF-α using computer modeling and deletion.mutant technique

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Science China(Life Sciences) 2004年第3期47卷 279-286页

作者： ZHANG Wei FENG Jiannan SHEN Beifen Beijing Basic Medical Institute Beijing 100850 China Beijing Basic Medical Institute Beijing 100850 China Beijing Basic Medical Institute Beijing 100850 China

The genes of the heavy and light chain variable region (VH, VL) of Z12 antibody against hTNF-a were cloned, and according to the translated sequence of amino acids, the spa-tial structures of VH and VL domains were modeled by using homology-based modeling method, followed by constructing the whole three-dimensional structure of Fv fragment. The complex model of Fv interacting with hTNF-a was gained with computer-guided molecular docking method, based on which, it was predicted that the epitope recognized by Z12 was from 141 to 146 of hTNF-a. hTNF-a molecule was divided into two fragments of N-terminal region from 1 to 91 and C-terminal region from 92 to 157 with prokaryotic expression. The measured results suggested that the antigenic epitope recognized by Z12 antibody was located in the C-terminal region 92-157 of hTNF-a, proving the predicted result reliable preliminarily. Further experimental results showed that after hTNF- 141-146 residues were deleted, Z12 antibody almost lost the ability to recognize the mutant, suggesting that the amino acid residues from 141 to 146 of hTNF-a were specially recognized by Z12 antibody.

关键词： human tumor necrosis factor-α, Fv, antigenic epitope, homology-based modeling, deletion.

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TRANSTHYRETIN (PREALBUMIN) GENE IN HUMAN PRIMARY HEPATIC CANCER

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Science China Chemistry 1991年第11期34卷 1312-1318页

作者：顾建人蒋惠秋贺丽萍李岱宗周筱梅戴卫列钱莲芳陈渊卿 C. SCHWEINFEST T. PAPAS Notional Laboratory for Oncogenes and Related Genes Shanghai Cancer InstituteShanghai 200032 PRC Laboratory of Molecular Oncology National Cancer Institute(NCI-FCRDC)USA

From a subtracting cDNA library constructed from normal liver versus human primary hepatic cancer (PHC), a cDNA clone pG8 was isolated. Using it as a probe, RNA extracted from one human liver and 9 PHC samples were analyzed by Northern hybridization. As expected, its mRNA was highly expressed in liver; however, the expression was strikingly suppressed in PHC. Only weak signal was observed in 2 out of 9 PHC, while no signal was detectable in the other 7 samples. Utilizing pG8 as a probe, DNA from the same PHC specimens was analyzed after MspI digestion and Southern hybridization. deletion.of DNA fragment was observed in 4 out of 9 samples. In further study of cancer and non-cancerous liver from other 7 PHC patients, similar deletion.of DNA fragments in cancer was observed in 4 out of 7 samples. After sequencing of the clone of 572 bp, it was unexpectedly found that pG8 was completely homologous to the coding sequence of transthyretin, TTR gene, as TTR (or prealbumin) gene has been known to be linked to a hereditary disorder, familial amyloidosis (FAP), and related to thyroxine transport and binding to retinol-RBP (the retinol binding protein) complex. This is the first report of a study on TTR in human primary hepatic cancer. Since TTR gene was strikingly suppressed in mRNA expression and possibly defective in its gene structure, it was strongly implicated that TTR might be an important gene marker or a candidate of anti-oncogene for human PHC. The biological activity of TTR gene is under study.

关键词： hepatoma subtracting cDNA library suppression deletion.

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