Cloning And Bioinformatics Analysis Of CXCL12 And CXCR4 Encoding Genes In Japanese Lamprey(lampetra Japonica)

Lamprey is the most primitive jawless vertebrate so far,and it is a real"living fossil".Cambrian ostracoderms and lamprey have a common ancestor.Lamprey is regarded as a new model organism of adaptive evolution and origin in both vertebrate evolution and immune system research.Sea lamprey?Petromyzon marinus?,as one of the typical representatives of jawless vertebrates,has become a model organism of Marine biological evolution.Japanese lamprey?Lampetra japonica?,a species closely related to sea lamprey,is distributed in the Yalu river basin in northeast China.Current basic research on lampreys focuses on Japanese lamprey and sea lamprey that have completed whole genome sequencing.In mammals,Chemokine?C-X-C motif?ligand 12?CXCL12?also known as stromal cell-derived factor-1?SDF-1?and pre-B cell growth-stimulating factor,and its CXC chemokine receptor 4?CXCR4?,respectively,belong to CXC chemokines and CXCR type G protein coupled receptor?GPCR?superfamily.Recent functional studies have shown that the CXCL12/CXCR4 axis plays an important role in multiple biological processes,such as immunity,inflammation,embryonic development,organogenesis,tumor,HIV disease,warts-hypogammaglobulinemia-infections,myelokathexis-syndrome?WHIM?,and has become one of the focuses of current biological research.However,whether CXCL12 and CXCR4 are ligand-receptor relationship in lamprey is unknown,and their interaction and immunological mechanism in lamprey's are rarely reported.Although the complete genome sequencing of Japanese lamprey has been completed,there are still many genes that are not clearly annotated compared with the integrity of the latest genome sequencing assembly of sea lamprey.For the first time,this study carried out information mining on the immune related CXCL12 gene and its possible receptor CXCR4 gene in Japanese lamprey by bioinformatics methods basing on the well-sequenced and well-assembled genome database of sea lamprey,combined with the genomic database information of Japanese lamprey.The results found that CXCL12 and CXCR4 homologous genes exist in the genome of Japanese lamprey.This led to further research into them as target genes.Firstly,Primers for CXCL12 sequence were designed and synthesized,from the source of Japanese lamprey nasal tissue cDNA of amplification and cloning of Japanese lamprey CXCL12 gene coding region,using polymerase chain reaction?PCR?technology for Japanese lamprey CXCL12 sequence,build pMD19-T-CXCL12recombinant plasmid sequencing,after confirmed the correctness of the sequence,and combined with bioinformatics methods for Japanese lamprey CXCL12 sequence analysis.It includes the open reading frame?ORF?,basic physical and chemical properties,hydrophilic/hydrophobic properties,transmembrane domain,functional domain,conserved structure and secondary structure of the protein were predicted and analyzed.Meanwhile,the CXCL12 protein was systematically analyzed by N-J algorithm.The results showed that the CXCL12 encoding region of Japanese lamprey was 267 bp and encoded 88 amino acids.The relative molecular weight was 10.10 kD,the theoretical isoelectric point was 10.60,and the net charge at pH=7 was 11.80.Among the amino acid sequences of CXCL12,the conserved C31 and C33 contain a proline residue and CXCL12 contains a signal peptide composed of 22 amino acids,and the four conserved cysteine residues are strongly conserved and belong to the typical CXC-type chemokine.This study provides a theoretical basis for further exploring the function of CXCL12 gene in lamprey immunology.Subsequently,Primers for CXCR4 sequence were designed and synthesized,from the source of Japanese lamprey nasal tissue cDNA of amplification and cloning of Japanese lamprey CXCR4 gene coding region,using polymerase chain reaction?PCR?technology for Japanese lamprey CXCR4 sequence,construction of Rho-pCMV-CXCR4 and Lucy-Rho-pCMV-CXCR4 eukaryotic expression vector sequencing,after confirm the correctness of the sequence,and combined with bioinformatics methods for Japanese lamprey CXCR4 sequence analysis.Meanwhile,the recombinant plasmid was transfected into HEK293T cells,and the expression of CXCR4 was detected by indirect immunofluorescence chemistry?ICC?after 24 hours.It includes the open reading frame?ORF?,basic physical and chemical properties,hydrophilic/hydrophobic properties,transmembrane structural domain,functional domain and secondary and tertiary structure of the protein were predicted and analyzed.Meanwhile,the CXCR4protein was systematically analyzed by N-J algorithm.The results showed that the CXCR4 encoding region of Japanese lamprey was 1113 bp and encoded 370 amino acids.The relative molecular weight was 40.90 kD,the theoretical isoelectric point was8.28,and the net charge at pH=7 was 4.30,the Japanese lamprey CXCR4 protein contains seven transmembrane domain structure,belongs to the family of GPCR,speculate Japanese lamprey CXCR4 protein function and may be consistent with other known mammal of CXCR4 protein function.At the same time,the prediction of the secondary structure and tertiary structure of CXCR4 protein in Japanese lamprey showed that the content of?-helix was the highest in the secondary structure,indicating that it had multiple transmembrane structures and was consistent with the characteristics of transmembrane protein.Random crimp accounted for about 30%,indicating that the conformational diversity of the protein was consistent with the conformational changes of the activated and closed Ca²⁺channel protein.The third-order structure?-helix region forms a hollow spiral cylinder,which provides a basis for the passage of ions.In addition,CXCR4 protein of lampreys japonicus in cells may play a role as an ion channel.Finally,the Japanese lamprey CXCR4 can successfully express in HEK293T cells using immunocytochemistry method.In summary,the chemokine CXCL12 and its possible receptor CXCR4 have been cloned,and their molecular structure and bioinformatics also analyzed.These results lay the important foundation for validation the subsequent ligand-receptor pair relationship between CXCL12 and CXCR4 in Japanese lamprey,at the same time,it will be help for Japanese lamprey CXCR4 protein function research and screening new drug targets and the treatment of diseases.