| Mammal immunoglobulin is four peptide chains molecule composed of two heavy chains and two light chains. The variable region (V) of heavy chains and light chains which consists of antigenic determinant is the specific site recognized and binded by antigen. The relatively constant FR with highly variant CDR, constitutes three dimensional complementary spatial structure on antigen surface. As effector fragment of immunoglobulin, C region of heavy chain has a important role for Ig function realization.Many mammal have five kinds of Ig molecule, IgM,IgD,IgG,IgE and IgA, coded byμ,δ,γ,ε,αgene, respectively. According to the difference of antigen specificity in C region of Ig light chain, it is also divided into lambda (λ) and kappa (κ)chains.Mammal Ig molecule is coded by IgK, IgL and IgH genes. A number of DNA fragments (gene segments) in germline are rearranged to translate into a polypeptide chain. In 1965, Dreyer and Bennet first introduce hypothesis, V region and C region of Ig are coded by separated genes.These two genes are rearranged together by translocation in the process of lymphocyte development. In 1976, Hozumi and Tonegawa confirm the hypothesis by DNA recombination technique.A large of V (D) and J gene segments constitute germline V genes in genome. V region is generated by different selection and rearrangement of these gene segments. Due to immune system phyletic and evolution analysis, V gene should be paid close attention. IgM is the first immunoglobulin and one of the important media in humoral immune response. Up to now, little papers has been published about dog Ig genetic characterization and V region feature, this experiment will provide molecular biological basis for understanding dog immune function and diverse antibody repertoire.To analyze dog IgM constant region for its bioinformatics and evolution, RT-PCR and 3′RACE methods were used to obtain IgM constant region sequences including secreted and transmembrane form tails. Bioinformatics and phylogeny analysis suggested that the structure of dog IgM showed typical mammalian IgM characteristics. Dog IGHM gene contains four exons separated by three introns and above 60% amino acid homology compared with other species. Comparison result from various structural domains demonstrated that homologies have the increase tendency towards molecular carboxyl terminus. Cladogram analysis shows that dog IgM possesed by far higher homology with mammal, especial Carnivora animals than birds and fish. The homology analyzed with nucleotide sequences from different species indicates that dog IgM, within the various animals studied, is the most closest to panda (89.6%) and cat (84.6%) in evolution.According to known gene informations of heavy chain constant region, 85 VH sequences were obtained with dog IgM, IgD, IgG, and IgA primers by 5′RACE technique. It is the biggest VH population until now and the most complete variable region genes in related research.On the basis of the standard definition of VH gene families (The sequences of two different VH genes sharing above 75% similarity belong to the same family), all VH sequences belong to two families, designated as dog VH1 family and VH2 family. VH1 family included 84 sequences, separated into three groups which are homologous to human VH3 family. VH2 family only contained single sequence. The result showed that V region selection is biased towards VH1 family in the VH-D-JH rearrangement. The VH families of many domestic animals only belong to single family but several VH families in human and mouse, which are different to that of dog which lies in intermediate status.Dog V_H1 and V_H2 families are homologous to human V_H3 and V_H1 family, respectively. V_H3 is the biggest family which presumed that V_H3 gene family should duplicate gradually in evolution.This is relatively common phenomenon in vertebrate. In addition, diversity index (DI) of dog VH region(FR1～FR3) is higher than other mammal studied. DI value (109.25) of V region inαchain (57aa) is the biggest. Mutation frequency or mutability is very high in some positions. Somatic hypermu- tation may contribute to dog heavy chain variable region diversity.Nucleotides and lengths of 83 D gene segments varied greatly. Average value/ variance of amino acid length in CDR3 was 12.2/10.1. Compared with other species studied, length of dog D gene segments is moderate. D, N, P nucleotides almost constitute entire CDR3. N nucleotides almost make up the entire 5′end of CDR3 by abundant GC bases. In addition, many palindrome sequences were found, indicated a relatively large contribution by P nucleotides. Diversifications in D gene segments indicate that they derive from different VH-D-JH segments recombinations83 J_H segments belong to 3 gene families, designated as JI, JII, JIII family. JI family is subdivided into JIa and JIb groups. Alignment results from EMBL database show that dog germline JH genes may include 6 families. So, different D gene segments show preference to these three J_H gene families.All in all, VH multi-families, D gene segments diversity, somatic hypermutation, N and P nucleotide insertions, and different V_H-D-J_H segments recombinations contribute to dog Ig heavy chain V region diversity.From dog infected by vanine distemper virus, 38 V_H sequences ofμchain show above 90% nucleotide identity to each other with few base substitutions. All sequences belong to dog V_H1 family. In the specific state, V_H gene selection is biased toward similar human VH3-23 and VH3-21 families. Many clones have identical VH or D segments, suggested that they all derive from the same rearrangement. The result showed antibody repertoire trend towards specificity at specific pathogen infection status.C and V region genes of Ig _λand _κlight chains had been cloned and the genetic characters of V genes were studied. The results showed that the major features of dog V_λsequences were lower similarity and much bases variation. 32 V_λdeterminants were acquired, designated as dog V_λ1~V_λ5 family, and similar to human V_λ1,V_λ2,V_λ3,V_λ8,V_λ4 family respectively. V_λ1 family, the largest family in V_λ, is composed of 26 members and biased in V_λ-J_λrearrangement. V_λ2, V_λ3, V_λ4, V_λ5 family contained 1, 1, 2, 2 member respectively. Amino acid number in leader region of different families is diverged. All V_λ1 members possesed 23 amino acid residues signal peptides except for clone Lv65, which included 19 aa residues the same as V_λ2～V_λ5 families with more base mutations. 32 J_λsegments were clustered into four families and J_λ1 family included 20 members. V_λ-J_λwas rearranged randomly and biased toward J_λ1 family. Multi-families V_λand more variation than VH might suggest that light chain V region seems to contribute significantly to the diversity.An analysis of 36 V_κsequences led to the identification of one V_κfamiliy, which is similar to human V_κ1 family. 20 hydrophobic amino acids constructed dog V_κfamiliy signal peptide in leader region. At the nucleotide level, all sequences in leader region have above 90% identity to each other with few base substitutions. Interestingly, V gene homology is higher in kappa chain than lambda chain. All J_κsequences in this experiment belong to four families.In V_κ-J_κrearrangement, the same V_κfamily used different J_κfamilies, but prefer to J_κ1 which had little mutation compared with J_λ. Phylogenic analysis among light chain V region gene sequences from different species showed that V_λfamilies possesed higher interspecies identity than intraspecies identity and dog Vλgenes have be diverged and formed different families before some vertebrate segregation, even reptile and mammal divergence. It also indicated that these family members have existed before generation of germline gene. However, conservative Vκfamilies are the result of gene duplication during evolution.IgA, IgG, and IgD expressed levels were detected in different tissues of normal and immune dogs by real-time PCR technique. Each of them displayed high expression in lymphatic organ, such as spleen and lymph node.As expected, IgG was highly expressed in major pathological tissues. Parvovirus causes severe kidney pathological changes. Respiratory system (lung) and urinary system (kidney) were infected by canine distemper virus. In immune state, IgG is higher expressed in lung and kidney than other inimmune tissues.Secret IgA is synthesized by plasma cell in tunica propria of respiratory tract and digestive tube. In this experiment, IgA showed higher expression in mucosa tissue and small intestine in normal dogs and higher expression in small intestine than other tissues in immune dogs. It is reported that rabies virus and canine distemper virus can invade gastrointestinal mucosa. In addition, IgA was higher expressed in lung of immune dog than normal dog. It is indicated that IgA is exclusive mucosa Ig。The result showed that IgD was expressed in all experimental tissues in normal dogs. And spleen and lymph node presented higher expression than other tissues. But this expression is lower than IgG and IgA in these two tissues. Under immune state, IgD expression will increase significantly in spleen and lymph node. Unexpected, IgD expression level changed little at both states which suggested that IgD didn't carry out the function of neutralization pathogen in diseased region.
|
PDF Full Text Request