Association Between S-lap And Apoptosis And Preparation Of Polyclonal Antibody Against S-lap Protein

s-lap, a novel gene expressed in the forepart apoptic porcine retinal pigment epithelial(RPE) cells induced by visible light, was cloned by mRNA differential display analysis in ourlab. The register number of s-lap in GenBank is AF509472. s-lap gene has an open readingframe and encodes a polypeptide of 101 amino acid. Since the mRNA of s-lap only can beexpressed in the damaged RPE cells induced by visible light, but didn't express in normalRPE cells, we proposed that s-lap is closely related with the damage of RPE cells caused byvisible light. However, the function and its mechanism of s-lap are still unclear.In present study, the eukaryotic expression vector of s-lap, pcDNA3.1/s-lap/GFP wasconstructed and the cellular location of s-lap was investigated by transfecting four differentmammalian cell lines with the recombinant plasmid. The results showed that s-lap proteindistributed in the whole cell but give priority to the nuclear, which indicated it has somefunction in the nuclear.As s-lap only expressed in the forepart apoptic RPE cells induced by visible light, wefurther investigated the relationship between overexpression of s-lap and apoptosis. Afteroverexpression of s-lap in 293T cells, the genome was extracted and examined byelectrophoresis, the result did not show the typical DNA ladder which is the marker ofapoptosis. Nevertheless, the cell cycle analysis for 293T cells overexpressed s-lap indicatethat s-lap/GFP can make a distinct change in cell cycle distribution of 293T cells, cellnumber in G2/M phase was increased dramatically. So we assume that s-lap gene mayparticipate in the apoptosis process via its effect on cell cycle.Preparation of antibody against s-lap protein is essential for the futher study such astissue distribution and function of s-lap. So the prokaryotic expression vectorpET-28a(+)/s-lap was constructed and expressed s-lap protein in the E.coli BL21 induced byIPTG, then the s-lap recombinant protein with poly-His tag in the N-terminal was purifiedby Ni²⁺ affinity chromatography. Polyclonal antibody against s-lap was generated byimmunizing BALB/c mice with purfied His-tagged s-lap protein, and its specificity wastested by western blotting, the result showed that the polyclonal antibody against s-lapprotein (1:400 diluted) had good immunoreactivity, it could bind the recombinant s-lapprotein specifically. Our s-lap antibody establishes a substantial foundation for furtherresearch on the relativity between s-lap and apoptosis occurrence and its molecularmechanism.