Prokaryotic Expression And Antibody Preparation Of Cotton Cytoplasmic Male Sterility Related Gene Orf160

Cotton Cytoplasmic Male Sterility?CMS?has been applied to the production of cotton hybrid seeds,but the mechanism of cotton CMS is still unclear.Previous studies have found that an unique open reading frame?ORF?:orf160,exists in the mitochondrial genome of the cotton harknessii CMS line.orf160 is transcribed in the cotton CMS line but not transcribed in the cotton normal fertile line.It is speculated that orf160 may be associated with cotton CMS.However,the expression of this ORF at the protein level is still unclear,and the corresponding antibody for research is lacking.Therefore,it is necessary to prepare the antibody of ORF160 protein for further exploring the difference of orf160 in protein expression level and to clarify the correlation between orf160 and cotton CMS.The gene orf160 was optimized and synthesised according to prokaryotic expression codon biasing and analyzed by bioinformatics software.The gene was amplified by PCR and introduced into the Rsr II and Not I restriction sites.The recombinant expression vector was constructed by double enzyme digestion.The protein ORF160 was expressed in E.Coli BL21?DE3?,and the expression format was identified.The expression conditions of the protein were optimized and the protein was purified by Ni²⁺affinity chromatography column.Western Blot method was used to detecte the protein with anti-His-tag antibodies.The murine monoclonal antibody was prepared by immunizing Balb/C mice with ORF160 as antigen,The titer and the specificity of the antibodies were determined and identified.The double antibodies sandwich method for detecting ORF160 was initially established.The gene orf160 is 480 bp,encodes 159 amino acids.The gene orf160 was ligated with the expression vector pET-B2M after double enzyme digestion.Sequence analysis showed that the recombinant expression vector orf160-pET-B2M was constructed successfully.The protein ORF160 was expressed in the form of inclusion body in E.coli BL21?DE3?,and the size of protein was in accordance with the expected protein molecular weight.The optimal expression conditions of the protein were:IPTG concentration of 0.1 mM at 30°C for 4 h.and the concentration of the protein purified by Ni²⁺affinity chromatography column was 1.1 mg/mL.Western Blot analysis showed that the ORF160 could specifically bind to the anti-His-tag antibody,this result indicated that the expression of the protein was successful.The murine monoclonal antibody was prepared using purified ORF160.Three hybridoma cell lines that could stably produced antibodies were selected:2B9?6 400×?,2G10?6 400×?and 3E9?3200×?.The experiments showed that all these antibodies had specificity to ORF160.An immunological method for the detection of ORF160 was successfully established with a detection limit of no more than 1.25?g/mL.This experiment successfully constructed the prokaryotic expression vector orf160-pET-B2M.ORF160 was expressed and purified.The anti-ORF160 murine monoclonal antibody was successfully prepared.And an immunological method to detect ORF160 was established.This study laid the foundation for further research on the correlation between orf160 and cotton CMS.