RESUMO
The major histocompatibility complex is a diverse gene family that plays a crucial role in the adaptive immune system. In humans, the MHC class I genes consist of the classical loci of HLA-A, -B, and -C, and the nonclassical loci HLA-E, -F, and -G. In Platyrrhini species, few MHC class I genes have been described so far and were classified as MHC-E, MHC-F, and MHC-G, with MHC-G possibly representing a classical MHC class I locus while there were arguments about the existence of the MHC-B locus in Platyrrhini. In this study, MHC class I genes were identified in eight common marmosets (Callithrix jacchus) and two brown-headed spider monkeys (Ateles fusciceps). For common marmosets, 401 cDNA sequences were sequenced and 18 alleles were detected, including 14 Caja-G alleles and 4 Caja-B alleles. Five to eleven Caja-G alleles and one to three Caja-B alleles were detected in each animal. For brown-headed spider monkeys, 102 cDNA sequences were analyzed, and 9 new alleles were identified, including 5 Atfu-G and 4 Atfu-B alleles. Two or three Atfu-G and two Atfu-B alleles were obtained for each of animal. In phylogenetic analyses, the MHC-G and -B alleles from the two species and other Platyrrhini species show locus-specific clusters with bootstrap values of 86% and 50%. The results of pairwise sequence comparisons and an excess of non-synonymous nucleotide substitutions in the PBR region are consistent with the suggestion that Caja-G and Atfu-G may be classical MHC class I loci in the Platyrrhini species But it appears that MHC-B locus of the two Platyrrhini species shares features with both classical and nonclasical MHC class I loci. Our results are an important addition to the limited MHC immunogenetic information available for the Platyrrhini species.
Assuntos
Atelinae/genética , Callithrix/genética , Genes MHC Classe I , Alelos , Sequência de Aminoácidos , Animais , Evolução Molecular , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
OBJECTIVE: To express and analyze p46000 antigen from newborn larvae of Trichinella spiralis. METHODS: p46000 antigen gene was subcloned to the pET28a expression system by PCR. The recombinant transformant was induced by IPTG and the antigenicity was analyzed with ELISA and Western blotting. RESULTS: The molecular weight of the expressed protein was about Mr 48000. ELISA and Western blotting showed that this recombinant protein could be recognized by T. spiralis infected swine serum and rabbit anti-recombinant protein serum, and the rabbit anti-recombinant protein serum could recognize a Mr 46000 protein from newborn larvae of T. spiralis. CONCLUSION: p46000 recombinant antigen from newborn larvae of T. spiralis was expressed which shows a specific antigenicity.
Assuntos
Antígenos de Helmintos/biossíntese , Trichinella spiralis/imunologia , Animais , Antígenos de Helmintos/imunologia , Expressão Gênica , Soros Imunes , Larva/imunologia , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , SuínosRESUMO
OBJECTIVE: To clone and characterize the p43 cDNA from muscle larvae cDNA library of Trichinella spiralis (Ts) Chinese isolate. METHODS: PCR technique was used to amplify the target cDNA from muscle larvae cDNA library. After cloned in pMD18T vector, it was transformed into E. coli NovaBlue. The positive clones were sequenced and the cDNA was cloned into pET28a expression vector. After induced by IPTG, the inclusion body of the recombinant protein was purified and re-natured. The deoxyribonuclease II (DNase II) activity of the recombinant protein was tested by hydrolyzing XDNA. RESULTS: Open reading frame (ORF) of the p43 cDNA was successfully cloned from the muscle larvae cDNA library of the Chinese Ts, there were mutations of two nucleotides in the ORF of the Chinese Ts p43 cDNA comparing with that from U.S.A. isolate at the positions 210 and 604, namely, C and A in the USA isolate but T and G in the Chinese isolate. Considering that three authors had cloned the same p43 cDNA from the USA isolate and six groups (including this team) had also obtained the same sequence from the Chinese isolate, the mutation of the two nucleotides was considered as the single nucleotide polymorphic (SNP) marker of the Chinese Ts isolate. The DNase II activity of the recombinant protein was successfully detected by hydrolyzing lamdaDNA. CONCLUSION: The p43 cDNA was successfully cloned from the muscle larvae cDNA library of the Ts Chinese isolate. Two SNPs were found in the nucleotide sequence. The DNase II activity was proved.
Assuntos
Endodesoxirribonucleases/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/metabolismo , Trichinella spiralis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Biblioteca Gênica , Glicoproteínas de Membrana/biossíntese , Dados de Sequência Molecular , Fases de Leitura Aberta , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/biossíntese , Proteínas Recombinantes de Fusão/biossínteseRESUMO
OBJECTIVE: To obtain antigenic genes encoding newborn larvae antigens of Trichinella spiralis. METHODS: Newborn larvae (NBL) cDNA library of Trichinella spiralis was screened using the infected swine serum and positive clones were sequenced and analysed. RESULTS: 158 positive clones were obtained from 4.5 x 10(5) recombinant clones by immunoscreening. The sequence analysis of positive clones showed that there are 36 novel cDNAs, 5 reported cDNAs and 12 cDNAs without the ORF showing high similarity to the mitochondrial DNA of Trichinella spiralis. CONCLUSION: Some cDNAs encoding antigenic protein of newborn larvae of Trichinella spiralis were obtained.
