Cloning Of Scavenger Receptor A Family And F Family Genes In Nibea Albiflora And Research On Related Immune Function

Yellow drum(Nibea albiflora)is the main economic fish caught in my country.Because of its delicious meat and high nutritional value,artificial breeding has been developed on a large scale.Yellow drum is often infected by pathogens of large yellow croaker and other adjacent net-caught fish during the cage culture process,which brings great harm to the culture process.Scavenger receptors(SRs)are a type of glycoprotein located on the surface of cells,which can recognize and take up low-density lipoprotein(LDL),and can participate in biological activities such as defense,cell adhesion,and phagocytosis of apoptotic cells.It plays an important role in the body’s natural immunity.In order to study the functions and interrelated mechanisms of the yellow drum scavenger receptor family genes in the immunity caused by pathogenic bacteria,this paper selected representative members of the yellow drum scavenger receptor family(A family and F family)for a detailed study.The full-length c DNA of the representative member of the yellow drum scavenger receptor family(NaSCARA3,NaSCARA4,NaSCARA5 and NaSCARF2)was cloned based on the transcriptome data of yellow drum,and related bioinformatics analysis was performed on it.The temporal and spatial expression regulation mode of the above-mentioned genes,the immune function of the recombinant protein,and subcellular localization were further analyzed.The main experimental results obtained are as follows.1.Based on the transcriptome data of yellow drum,the full-length c DNAs of the A family(NaSCARA3/NaSCARA4/NaSCARA5)and the F family(NaSCARF2)were obtained by PCR cloning.The full length of NaSCARA3 c DNA was 2562 bp,the open reading frame(ORF)was 1821 bp,encoding 606 amino acids;the full length of NaSCARA4 c DNA was 4050 bp,the open reading frame(ORF)was 2382 bp,encoding793 amino acids;the full length of NaSCARA5 c DNA was 6968 bp,the open reading frame(ORF)was 1494 bp,encoding 497 amino acids;the full length of NaSCARF2 c DNA was 6540 bp,the open reading frame(ORF)was 2847 bp,encoding 948 amino acids.Clustal W analysis showed that the homology between NaSCARA3 and SCARA3 of other species were 73-89%,NaSCARA4 were 78-94%,NaSCARA5 were 87-97%,and NaSCARF2 were 92-93%.The CLECT(CRD)domain was found in NaSCARA4,and 6cysteine-rich regions were found in both NaCARA4 and NaCARA5 carboxyl terminus.Also found in NaSCARA3 and NaSCARA5 were TRAF2 binding domains and tyrosine conserved regions involved in internal folding.The NaSCARF2 molecule contains 5 EGF and 3 EGF-like domains.2.The results of tissue differences showed that the genes of the yellow drum scavenger receptor family A and F were distributed in the 7 tissues tested(spleen,liver,kidney,gill,stomach,intestine and heart),among which the A family gene expression was the highest in the spleen or liver.NaSCARA3 and NaSCARA5 were the highest in the spleen,which were 67.16 and 24.42-fold higher than those in the control group(heart).The NaSCARA4 expression was the highest in the liver,47.29-fold that of the control group(heart).The highest expression in the liver of yellow drum was the F family(NaSCARF1 and NaSCARF2),which were 19.86 and 45.13-fold that of the control group(heart),respectively.All detected genes showed time-dependent up-regulation of pathogenic Vibrio(Vibrio alginolyticus,Vibrio parahaemolyticus)infection or Poly I:C stimulation,but the time to the highest expression level was slightly different,most of them were concentrated between 8-36 h.Combined with the above experimental results,it is found that the A family and the F family have a strong stress response to pathogen infection,but the specific regulation mechanism needs further study.3.Using p ET-32 a as the vector and E.coli BL21(DE3)as the host bacteria,the prokaryotic expression vectors p ET32a-NaSCARA3,p ET32a-NaSCARA4＿CRD,p ET32a-NaSCARA5 and p ET32a-NaSCARF2＿EGF were successfully constructed.The IPTG induction concentration and induction time were optimized,and the purification process was established.Purified by the Ni-NAT Superflow resin kit,a single high-purity recombinant protein was obtained.After renaturation,it was used for in vitro bacterial binding experiments.After Western blotting,it was found that the recombinant protein could bind to all the tested bacteria in the experiment(Vibrio alginolyticus,Vibrio parahaemolyticus and Vibrio harveyi).The above experimental results ensure the supply of materials for more in-depth research on the function of scavenger receptor molecules,and also provide important key technical support for the fermentation production of fish genetic engineering products,and the development of innovative drugs or additives.4.The eukaryotic expression vector of the NaSCARA5-EGFP fusion protein was constructed and transferred into carp epithelial cells(EPC).Laser confocal microscopy showed that NaSCARA5 was mainly expressed in the cell membrane,indicating that NaSCARA5 was a typical transmembrane protein.It is speculated that this molecule may be related to signal recognition and conduction inside and outside the cell or inside and outside the organelle.The above results fully indicate that the gene members of the scavenger receptor family of yellow drum are involved in the body’s natural immune response as important membrane proteins.The research results will help to understand the role of scavenger receptors in the innate immune receptor family of sciaenidae,which can provide reference for the prevention and treatment of fish diseases and the research of vaccines.