Germline Gene Locus Structure And Diversity Mechanism Exploration Of Goat Immunoglobulin Heavy Chain Variable Region

Immunoglobulin（Ig）is an important molecule in adaptive immune response.The typical immunoglobulin molecule consists of two light chains,namely,λchain andκchain.Currently,most related studies have focused on humans,mice and some large artiodactyls,but there is still a gap in goats’light chains.As we know goats has wide range of food intake and its immunity is relatively high.The research for structure of goat immunoglobulin gene and diversity mechanism is helpful to reveal the molecular mechanism of the animals’disease resistance,so as to lay a foundation to improve the immunity of animals and humans.We cloned variable region of goat immunoglobulin light chain by 5’RACE in this experiment,and then analysed the structure and diversity of germline genes to uncover expression characteristics based on the goat genome database completed recently.The results are as following:（1）The results showedλlight chain variable region genes contained at least 35 V_λgene fragments which were divided into seven potential functional genes,one ORF and 27pseudogenes.Seven potential functional genes,1 ORF1 and 25 pseudogenes got together defined as the first family;p-V2 and p-V27 belonged to the second gene family.Three（J_λ-C_λ）s are arranged in the downstream with a pseudogene J_λ3.There are five potential functional genes,two ORF and four pseudogene fragments in theκlight chain,which are divided into three gene families according to similarity.Three J_κgenes are located in the upstream of a C_κ.Gene locus structure inλlight chain is V_λ（35）-J_λ3-C_λ3-J_λ2-C_λ1-J_λ1-C_λ2,and that is V_κ（35）-J_κ（3）-C_κinκlight chain.（2）305 unduplicated cDNA sequences inλchain and 169 cDNA sequences inκchain were amplified from the spleen of 3 Xinong Saanen Dairy Goats using 5’RACE.On expression level,the preference for use of V segments in VJ recombination is found.λchain mainly expressed V_λ2（26.23%）and V_λ3（73.11%）,in addition,κchain mainly expressed predominate V_κ2（52.07%）and V_κ4（46.15%）.In terms of the number of N+P nucleotides and the length of CDR3 amino acids,the junction diversity of theλchain is more abundant than that of theκchain.（3）The single base substitution mutation and insertion/deletion of the cDNA sequences inλchain andκchain were analyzed,and characteristics of somatic hypermutation were also analyzed.We found C→T and A→G mutations predominated inλchain andκchain with mutation in A:T base pairs more than mutation in the G:C base pairs,so base substitution mutations tended to occur in the pyrimidine nucleotide.Base transversion mutations were more than convert mutations.Overall mutations occur more frequently in CDRs than FRs.The frequency of base insertions and deletions inλchain is also higher than theκchain.This study focused on the gene structure of goat immunoglobulin light chain and the important role of VJ recombination and somatic hypermutation mechanism on the diversity of light chain variable region.