Cloning,Prokaryotic Expression And Polyclonal Antibody Preparation Of Lymphocyte Antigen 75 In Oreochromis Niloticus

Lymphocyte antigen 75(Ly75)is a type I transmembrane surface protein that belongs to the mannose receptor family of C-type lectin endocytic uptake receptors.Studies have shown that Ly75 is highly expressed in gonads of fish and specifically expressed in some specific germ cells.In order to investigate the function of Ly75 in Nile tilapia(Oreochromis niloticus),we isolated the full c DNA sequence of On Ly75 by RACE approach in Nile tilapia.The tissue distribution of the gene in healthy Nile tilapia was analyzed by q PCR.The prokaryotic expression vector of On Ly75 was constructed,and the On Ly75 recombinant protein was successfully obtained.The purified On Ly75 recombinant protein was used to immunize New Zealand white rabbits to obtain the rabbit anti-On Ly75 polyclonal antibody.In addition,the m RNA expression distribution of On Ly75 in the testis tissue of Nile tilapia was studied by in situ hybridization.The main results were as follows: 1.Molecular cloning and expression of Ly75 gene in Nile tilapiaThe primers were designed by using the predicted sequence of On Ly75 on NCBI as template.The full sequence of On Ly75 gene was cloned by RACE technique.The results showed that the 6632 bp full length of On Ly75 c DNA sequence was successfully obtained,with an open reading frame(ORF)of 5199 bp,encoding 1732 amino acid residues.The physicochemical properties of On Ly75 protein were analyzed by Prot Param software,the results showed that the molecular weight of the protein was196.5 kilo Daltons,the theoretical isoelectric point was6.41,the instability index was 38.17 andthe instability index was 38.17,which means that the On Ly75 protein belonged to the stable hydrophilic protein;the grand average of hydropathicity was-0.476,which belonged to the hydrophilic protein.SMART software was used to analyze the conserved domain of On Ly75 protein,the conserved domains from N-terminal to C-terminal were as following: a Cys R,a FNII,ten CTLDs and a TM.The amino acid sequence homology analysis showed that On Ly75 had the highest homology with tuna Ly75(86.0% similarity,64.9% identity),and had high homology with other fish Ly75(75.3%~85.0% similarity,49.3%~62.3% identity).Phylogenetic tree analysis of NJ showed that On Ly75 gene belonged to the Ly75 protein subfamily of mannose receptor proteins,and showed strong associations with vertebrate Ly75 proteins.The expression of On Ly75 gene in healthy Nile tilapia was analyzed by real-time fluorescence quantitative PCR.The results showed that On Ly75 was expressed in all the tissues tested and the difference of their expression rate was significant.Among them,the expression rate in the testis was the highest,followed by that in intestine,thymus,posterior kidney,gill,spleen,head kidney,middle kidney and brain;the lower level was detected in the heart,muscle and ovary;the lowest level was detected in the skin and liver.According to the On Ly75 sequence of c DNA,primers were designed to prepare the Ly75 sense probe and Ly75 antisense probe for in situ hybridization.In situ hybridization was used to analyze the location of On Ly75 in the testis.2.Prokaryotic expression and preparation of polyclonal antibody of Ly75 in Nile tilapiaThe primers of the On Ly75 prokaryotic expression were designed by antigenicity peptide sequence.The On Ly75 was amplified using the c DNA of 8.5 days Nile tilapia embryoas template,and then was inserted into the prokaryotic expression vector p EASY-E2 to construct the the recombinant expression plasmid p EASY-E2-On Ly75.The recombinant plasmid was transformed into E.coli BL21(DE3)and induced by IPTG.The induced expression conditions was under the induction with 0.5 mmol/L IPTG for 5 h in 37℃.The On Ly75 recombinant protein was detected by SDS-PAGE.The result showed that the On Ly75 recombinant protein was mainly found in inclusion bodies.The purified On Ly75 recombinant protein was used to immunize New Zealand white rabbits for several times,and the antiserum was obtained.The rabbit anti-On Ly75 polyclonal antibody was obtained by affinity purification.The polyclonal antibody was tested by Western blot in different organs of Nile tilapia.The results showed that the polyclonal antibody was highly expressed in the testis,indicating that On Ly75 polyclonal antibody was successfully prepared.