Prokaryotic Expression Of NYD-SP15 And The Preparation Of Polyclonal Antibody As Well As Molecular Cloning And Characterization Of Lims E: A Development Related Gene

Objective:1, To construct the prokaryotic expression plasmid of NYD-SP15which is related with proliferating lesion, induce protein expression invitro and immunize mouse for antibody.and use the polyconal antibodyfor the immunohistochemical staining of the proliferativevitreoretinopathy.2, To study the role of different splicing variant of LIM andsenescent cell antigen-like domains Lims gene relative to celldevelopment.Methods:1, NYD-SP15 gene was amplified using PCR technique, theninserted into PET28a prokaryotic expression vector. After genesequencing of the recombinant plasmid, PET28a-NYD-SP15 wastransformed into expressing strain, its protein expression was induced by propyl-β-D-thiogalactosides (isoproprlthiogal actoside IPTG), thenpurified by Ni stele. 8- week- aged BALB/C mice were immunized by thepurified protein to obtain antibody. The protein and its antibody werechecked by western-blot technique, utilize the immunohistochemicalmethods to study the clinical sample for the proliferativevitreoretinopathy.2, Splicing variants of Lims gene were amplified by RT-PCR frommouse cDNA and inserted into Pinpoint-T vector, two position clonesselected by PCR were sequence.Results:1, We have successfully amplified NYD-SP15 gene by PCRtechnique, obtained highly NYD-SP15' expressive strain and the Histagged-fusion protein which molecular weight was about 58 kilodalton(KD). analyzing by western-blot, the polyclonal antibody of the proteinprepared by immunizing mice, is specific antibody for NYD-SP15. Theimmunohistochemical staining display, the NYD-SP15 gene located inthe plasm of RPE, showing brown dyeing.2, Lims E, a novel splicing variant with a 1164 bp open readingframe (ORF), encoding a 387-amino acid (AA) protein was cloned.Conclusion:1, NYD-SP15 prokaryotic expression vector, and the highlyexpression protein in vitro as well as its polyconal antibody, the expression of NYD-SP15 gene in retinal proliferative membranes, thesewill establish a substantial foundation for ocular proliferations, especiallyfor the proliferative retinopathy.2, Comparative genome analysis displayed Lims E, a novel variantof Lims gene was confirmed in mouse and established the foundation forfurther to studying the function of Lims E in cell development.