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Objective:To clone the cDNA in full-length of human mammaglobin,do prokaryotic expression and purify hMAM protein,as a basis for early diagnosis of breast cancer.Methods :hMAM cDNA was amplified through RT-PCR from breast cancer tissue and breast cancer cell line MD-MB453,and the recombination pQE40-hMAM vector was constructed and expressed in E.Coli.M15 after induction by IPTG.The fusion protein was purified with Ni-NTA-His affinity chromatography. Results: Two subtypes of hMAM cDNA and the hMAM(Isoform) cDNA were found,in which consisted of 270 bp,different from the wildtype hMAM cDNA of 279 bp on nine continuous base pair missing.The fusion protein formed inclusion body in prokaryotic expression system and the renatured protein was purified which purity was about 97%.Conclusion: The recombinant hMAM was successfully purified.
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Objective To construct a single chain Fv immunotoxin HAb25(scFv)-PE40 and explore its targeting effects on human hepatocellular carcinoma(HCC) cells.Methods First,the HAb25(scFv) gene was ligated with PE40,a truncated form of pseudomonas exotoxin on the the plasmid vector ply5 to form the HAb25(scFv)-PE40 gene.Then the HAb25(scFv)-PE40 gene was subcloned into the pBV220 E.Coli expression vector for production of the recombinant immunotoxin.SDS-PAGE was used to identify the expression of objective protein.The specificity of HAb25(scFv)-PE40 binding to HCC cells was tested by indirect immunofluorescent staining,and the in vitro targeting cytotoxic assay was employed to observe the selective cytotoxic activity of the objective protein.Results Enzyme digestion and sequencing analysis proved the construction of HAb25(scFv)-PE40 gene in ply5 vector.HAb25(scFv)-PE40 gene was highly expressed in pBV220 vector(up to 43.3% of total cellular proteins).Indirect immunofluorescent staining showed HAb25(scFv)-PE40 could specifically bind to HCC cells as its parent antibody HAb25.The protein was verified to have selective cytotoxic activity when cocultured with HCC cells.Conclusion A single chain Fv immunotoxin HAb25(scFv)-PE40 was successfully constructed.The immunotoxin has selective cytotoxic activity against HCC,which found a basis for further study on its basic and clinical application.
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Macrophage migration inhibitory factor(MIF),a potential pro inflammatory cytokine,is a pivotal regulatory element in the nature and adapted immune.This article aims to introduce the histocellular sources,genetic structure,protein structure and biological activity of this cytokine,and further to explore its role in the pathogenesis of septic shock,and its antagonist in the treatment of this disease.
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In order to assess the significance of human malignancy-associated nucleolar antigen P24 in the diagnosis of tumors,552 specimens of human tissues were stained and observed with immuno - fluorescent and ABC methods using anti-P24 monoclonal antibody MA2.It was found that P24 was positive in the nucleolus of 97% cases of malignant tumors and in 50% of atypical hyperplastic tissues but negative in 98.6% cases of benign tumors and in all the normal or nontumorous tissues.On the basis of these findings,it is believed that P24 is highly specific to malignant tumors and will be a useful marker for the differential diagnosis between malignant and benign tumors.