Preparation Of Polyclonal Antibody Of Transport Protein IFT25 And DYF-1 In The Cilia Of Chlamydomonas Reinhardtii

In order to maintain the normal function of cilia needs synergy of various ciliary transport proteins within the cilia,in recent years various transport proteins within the cilia function studies and reports more and more.IFT25 and IFT70(DYF-1)are IFT-B composite components,transport proteins involved in cilia inside,but few studies have reported both in the Chlamydomonas reinhardtii.Research the function of Chlamydomonas reinhardtii IFT25 and IFT70(DYF-1)is dependent on the two corresponding antibody,the experimental tool.This experiment attempts to prepare polyclonal antibodies of IFT25 and IFT70(DYF-1),and to detect its specific use Western Bloting.First,the gene fragment was amplified out by PCR from the plasmid containing the target genes(due to longer full dyf-1 gene,only the amplified N 'partial fragment),and were constructed to pMAL-c2X,pGEX-2T and pET-28a(+)expression vectors respectively,ultimately,six kinds of,plasmid pMAL-c2X-ift25,pGEX-2T-ift25,pET-28a(+)-ift25 and pM AL-c2X-dy f-1(3 80),pGEX-2T-dy f-1(380),pET-28a(+)-dyf-1(380),ware obtained.The six plasmids were transformed into E.coli BL21(DE3),which was broken by ultrasonic,induced with IPTG and Analysis of soluble fusion proteins respectively by SDS-PAGE,the result are that fusion protein MBP-IFT25 was water soluble,only part of the GST-IFT25 water-soluble,6×His-IFT25 was water-insoluble and MBP-DYF-1(380)was water-soluble,GST-DYF-1(380)was water-insoluble,6×His-DYF-1(380)was water-insoluble.Since the fusion protein MBP-IFT25 and MBP-DYF-1(380)was water-soluble form,and purified by affinity purification methods under non-denaturing conditions;6 × His-IFT25 and 6 × His-DYF-1(380)was water-insoluble and affinity purified under denaturing conditions.The results were all obtainedhigh purity of fusion protein.water-solublefusion protein MBP-IFT25 and MBP-DYF-1(380)as antigen to immune rabbits with freund's adjuvant emulsified to prepare polyclonal antibody.After boosting 3-4 times,the titer of antisera was measured by ELISA,When the antiserum titer of more than 100,000,the separation of the antisera.Results After the fifth immunization,the titers of both two antisera were more than 128,000,which can meet the test requirements.Protein A can specificity combine with all IgG within antisera.After Protein A purification,the peak concentration of two antisera reached 13 mg/mL(a-IFT25)and 10 mg/mL(a-DYF-1)respectively.The antibodies after Protein A purification and nitrocellulose membrane purification method,in a dilution of 1:1000 condition can be correctly bind to target protein in Chlamydomonas reinhardtii through Western Bloting.The purified antibodies with high specificity,can be used for subsequent expriments.