Analysis Of Antigenic Epitopes And Establishment Of Immunoassay Method For RecQL4

The DNA double helix was proposed by Wason and Crick, DNA polymerase wasdiscovered by people followed, it formed a new chain from a single-stranded as templatedepend on DNA polymerase. Then, the scientists found another kind of enzyme which coulddestroy the hydrogen bonding of complementary base pairs in DNA replication, it couldopend DNA double helix, this kind of enzyme was named helicase by scientists. There arehundreds of helicase had been discovered. helicase is divided into a few families to research,according to its two-dimensional structure and level of sequence. Helicase has a variety offunctions after many years of research, they take part in DNA replication,DNA repair,transcription and RNA splicing, assassembly of ribosome, protein translation, stabilizetelomeres. Resently, some helicases were founded play an important role in the innateimmune reaction. Helicase has not only separate double-stranded nucleic acid structure ofenzyme, its biological function is far more than that.RecQ helicase family of the second family in helicase played a key role in the process innucleic acid metabolism. Recql1, WRN, BLM, Recql4andRecql5, in mammals contains fivemembers: Recql1, WRN, BLM, Recql4and Recql5, WRN, BLM and Recql4mutations canlead to three disease of autosomal-recessive that bloom Syndrome, Werner Syndrome andRots hmund Thomson Syndrome.Especially recql4as a member of the family of RecQ was founded it has a dual role inclinic, namely not only its function lack to promote a high incidence of tumor, and itsexceptionally high expression also can promote the progress of human tumor. Studies havefound that an abnormally high levels of RecQL4is closely related to the prostate tumor cellstumorigenicity. So, recql4is of important clinical value as anticancer drug development andthe new strategy of tumor treatment. This study expect analysis recql4epitopes and thedevelopment of specific McAb has been based and develop the related testing kits withpreliminary. For further research of correlation in RecQL4expression and tumor cells, andtumor of platinum or other kinds of chemotherapeutic drugs sensitivity related to lay thefoundation. This item using bioinformatics methods for recql4polypeptide antigen separately, andpredicted by the software, we identify five antigen epitope peptide sequence of amino acidsand synthetic. The purity of polypeptide epitope meet the requirement by identifying of massspectrometry and the purification by HPLC. And form the immune antigen by the synthesis ofpeptides and carrier protein KLH. Every polypeptide acquired a monoclonal antibody by thisantigen to immune Balb/c mouse. There are5hybridoma cell lines that is able to secreteantibody steadily as number1B5、2D6、3C8、4D2、5A7. Western blot shows that2antibodieswas combine with antigen expressed after affinity purification.The valence of antibody of2monoclonal antibody is1:1.24×105、1:1.24×105,respectively.the affinity constant isK(2D6)=2.1×109L/moL，K(5A7)=6.7×108L/moL respectively.We use the antibody of2D6and5A7to discussed the method of colloidal gold doubleantibody sandwich in course of colloidal gold technique. We can be sure the colloidal goldantibody of2D6as PAIgG, its concentration is4.8ug/ml,5A7as Cross membrane antibodies,its concentration is0.6mg/ml.We ensure the detecting range is0.25ng/ml~8ng/ml, linearequation is y=1.0356x+0.0039，R2=0.9924.