Characterization Of MHC Class Ⅱ Genes And Their Effects On Intestinal Parasitic Infection In White-Headed Langur

Major histocompatibility complex(MHC)is a kind of multi-gene family with high polymorphism in vertebrates,which is closely related to animal immune function,sexual selection and genetic evolution.MHC is often used as functional molecular marker to study the genetic diversity of species and to evaluate pathogen resistance.Compared with neutral molecular markers such as microsatellites and mitochondrial control region,it can also reflect the environmental adaptability and survival potential of species.The white-headed langur is a national class protected animal,and it is also a unique primate in China.Understanding its genetic diversity and survival potential is beneficial for formulating scientific and reasonable protection measures.In this study,the MHC class Ⅱ gene exon 2 of white-headed langur is obtained by polymerase chain reaction(PCR)and cloning sequencing.Characterization of MHC class Ⅱ genes are analyzed by bioinformatics software.At the same time,the infection of Amoeba and Blastocystis in white-headed langur are detected by molecular biological methods.Based on this,the effects of MHC class Ⅱ genes on intestinal parasite infection in white-headed langur is explored,thus providing a theoretical basis for the prevention and control of intestinal parasites in white-headed langur.The main research results are as follows:(1)The 136 effective sequences are identified as 22 Trle-DPB1 alleles,6 Trle-DQB1 alleles and 1 Trle-DQA1 allele.(2)There are 37 single nucleotide polymorphism sites in Trle-DPB1 alleles,resulting in27 amino acid changes.Among them,Trle-DPB1*21 has the most variation sites with 20.The amino acid sequences of Trle-DPB1 exon 2 are different,indicating that their variation sites are mainly non-synonymous substitution.There are 6 single nucleotide polymorphism sites in Trle-DQB1 alleles,resulting in 4 amino acid changes.Among them,Trle-DQB1*05 has the most variation sites with 4.The amino acid sequences of Trle-DQB1*02,Trle-DQB1*03 and Trle-DQB1*04 are the same,indicating that their variation sites are mainly synonymous substitution.In addition,the polymorphism information content and expected heterozygosity of the two genetic loci are both greater than 0.5,indicating that Trle-DPB1 and Trle-DQB1 have high polymorphism.(3)The phylogenetic trees of MHC class Ⅱ gene exon 2 are constructed by neighbor-joining method and maximum likelihood method,respectively.The results show that no trans-species polymorphism in the MHC class Ⅱ genes of the white-headed langur in this study.In addition,in the selection pressure analysis,it finds that Trle-DPB1 is subjected to positive selection,while Trle-DQB1 is subjected to purification selection.(4)Molecular biological techniques are used to detect the parasitic infection of white-headed langur and identify the infected species.The result shows that white-headed langurs are infected with Entamoeba sp.RL3 and Blastocystis sp.subtype 13,and the infection rates are 91.67% and 25.00%,respectively.(5)SPSS 27.0 is used to analyze the correlation between MHC class Ⅱ exon 2 and protozoa infection.The results show that Trle-DPB1*21 and Trle-DQB1*05 are significantly negatively correlated with Amoeba infection,Trle-DQB1*01 allele is positively correlated with the infection of Blastocystis.There is no significant correlation between heterozygote and parasitic infection.In conclusion,the genetic polymorphisms of Trle-DPB1 and Trle-DQB1 are relatively high,and they are correlated with the infection of Amoeba and Blastocystis.It is preliminarily inferred that Trle-DPB1*21 and Trle-DQB1*05 may be involved in the recognition and presentation process of Amoeba antigen peptides,and Trle-DQB1*01 may be a susceptibility gene of Blastocystis.