To elucidate the structure of SLA-3 alleles on inbred line of Wuzhishan pig （WZSP） population, we examined the partial exon 1, completed exon 2, and partial exon 3 of SLA-3 loci using the reverse transcription-polym...To elucidate the structure of SLA-3 alleles on inbred line of Wuzhishan pig （WZSP） population, we examined the partial exon 1, completed exon 2, and partial exon 3 of SLA-3 loci using the reverse transcription-polymerase chain reaction （RT- PCR） and the sequencing-based method in 32 WZSPs. According to pedigree and amplification results, PCR products of 8 WZSPs were selected to clone and sequence. Nine different nucleotide sequences were obtained. After comparing the DNA and protein sequences of the WZSPs SLA-3 alleles with the published GenBank SLA sequences, it was found that the SLA-3 alleles in WZSPs were all novel, but there were very few variations among them. Comparision of SLA-3 and HLA-A protein sequences indicated that there was more sequence homology. Meanwhile, the construction of a phylogenetic tree using the nucleotide sequences of 23 SLA-3 alleles and 1 HLA-A allele represented that the WZSP population owns its unique genetics resource. In this study, the alleles of SLA-3 on WZSP group were successfully detected and analyzed, which provided the firm basis on the genotype of SLA-3 for breeding specific haplotypes WZSPs.展开更多
In order to obtain the TLR 4 gene sequence of Hainan Wuzhishan pig and analyze its molecular structure characteristics,its genomic sequence was cloned and sequenced by PCR and subjected to related bioinformatics analy...In order to obtain the TLR 4 gene sequence of Hainan Wuzhishan pig and analyze its molecular structure characteristics,its genomic sequence was cloned and sequenced by PCR and subjected to related bioinformatics analysis with the genomic sequence of the TLR 4 gene of pig in GenBank as a reference sequence.The full-length DNA sequence of Wuzhishan pig TLR 4 gene was 10 435 bp in length,of which CDS was 2 526 bp in length and encoded 841 amino acids.Compared with the common pig TLR 4 gene sequence published by GenBank,there were 38 mutations,of which two mutations were in the CDS region.The results of homology analysis showed that the amino acid sequence of the TLR 4 gene of Wuzhishan pig shared 99.9%,80.7%,79.9%,74.8%,74.7%,73.1%,73.0%,72.7%,62.5% and 43.4% homology with those of common pig,cattle,sheep,dog,horse,macaque,human,chimpanzee,mouse and chicken,respectively.The protein encoded by the TLR 4 of Wuzhishan pig belonged to secreted protein and transmembrane protein.It was hydrophilic,and had 8 glycosylation modification sites,17 serines (ser),7 threonines (Thr) and 8 tyrosine (TYR) phosphorylation sites.The amino acid phylogenetic tree analysis showed that the TLR 4 genes of different species are highly conserved during evolution.This result lays a foundation for further study of the function of TLR 4 gene.展开更多
[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of ...[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.展开更多
[ Objective] The study aimed to lay a foundation for studying immune recognition mechanism in xenotransplantation. [ Method] SLA- DQA gene of Hainan Wuzhishan miniature pig was cloned by RT-PCR and sequenced. J-Result...[ Objective] The study aimed to lay a foundation for studying immune recognition mechanism in xenotransplantation. [ Method] SLA- DQA gene of Hainan Wuzhishan miniature pig was cloned by RT-PCR and sequenced. J-Result] The SLA-DQA gene sequence of Wuzhishan minia- ture pig contained 768 nucleotides, among which 1 -765 were the complete open reading frame, encoding 255 amino acids. SLA-DQA cDNA se- quence and its deduced amino acid sequence of Wuzhishan miniature pig were most similar to those of the other miniature pigs in China. When compared with corresponding cDNA sequences and the amino acid sequences in other species, the homology with human was obviously higher than that with mouse. [ Conclusion] As the Chinese pig breed suitable for xenotransplantation, Wuzhishan miniature pig has significant importance to ser- ial studies on immunogenetics characteristics.展开更多
Skin grafting has been used as one of the most reliable tests to determine the genetic stability of laboratory animal such as mice and rats inbred line, but no identification of swine inbred lines by skin grafting has...Skin grafting has been used as one of the most reliable tests to determine the genetic stability of laboratory animal such as mice and rats inbred line, but no identification of swine inbred lines by skin grafting has been reported. At present, Wuzhishan miniature pig （WZSP） inbred line has acquired the F24 individuals in China. In order to verify whether WZSP inbred line had D^en cultivated successfully, allogeneic skin grafts and related research were performed on F20 individuals of WZSP inbreeding population, compared with a control group of autologous transplantation. We observed the transplant recipients＇ wounds, detected peripheral blood-related indicators interleukin-2, 4 and 10, CD4~ and CD8~ lymphocytes, and conducted hematoxylin-eosin （HE） and Masson＇s staining of skin to judge whether the immune rejection reactions occurred within 28 days after transplantation. Chr. 7 genomic heterozygosity of 48 WZSP individuals from F20 to F22 was analyzed by high-density single nucleotide polymorphism （SNP） chips （60 000 SNPs）. The result showed that there were no significant differences in graft skin, the plasma interleukin-2, 4, 10, CD4~ and CD8~, HE and Masson＇s staining results between the allograft and autograft groups, and no immune rejection occurred on the allograft group. We found that 11 genes in Chr. 7 of major histocompatibility complex （MHC） I and MHC II were homozygous which confirmed that immune antibody of the allograft and autograft groups were highly identical and also provided a theoretical basis to no immune rejection occurred on the allograft in the inbred WZSP. The result proved that the WZSP inbred line had been cultivated successfully for the first time in the world. The test methods also provide a scientific basis for the identification of swine and mammal inbred lines.展开更多
文摘To elucidate the structure of SLA-3 alleles on inbred line of Wuzhishan pig （WZSP） population, we examined the partial exon 1, completed exon 2, and partial exon 3 of SLA-3 loci using the reverse transcription-polymerase chain reaction （RT- PCR） and the sequencing-based method in 32 WZSPs. According to pedigree and amplification results, PCR products of 8 WZSPs were selected to clone and sequence. Nine different nucleotide sequences were obtained. After comparing the DNA and protein sequences of the WZSPs SLA-3 alleles with the published GenBank SLA sequences, it was found that the SLA-3 alleles in WZSPs were all novel, but there were very few variations among them. Comparision of SLA-3 and HLA-A protein sequences indicated that there was more sequence homology. Meanwhile, the construction of a phylogenetic tree using the nucleotide sequences of 23 SLA-3 alleles and 1 HLA-A allele represented that the WZSP population owns its unique genetics resource. In this study, the alleles of SLA-3 on WZSP group were successfully detected and analyzed, which provided the firm basis on the genotype of SLA-3 for breeding specific haplotypes WZSPs.
基金Supported by the Innovative Research Team Project of Natural Science Foundation of Hainan Province(2018CXTD345)Local Science Foundation of National Natural Science Foundation(31560696)+1 种基金Natural Science Foundation of Hainan Province(20153080)Special Fund for the Development of Local Science and Technology Guided by Central Government(ZY2019HN01)
文摘In order to obtain the TLR 4 gene sequence of Hainan Wuzhishan pig and analyze its molecular structure characteristics,its genomic sequence was cloned and sequenced by PCR and subjected to related bioinformatics analysis with the genomic sequence of the TLR 4 gene of pig in GenBank as a reference sequence.The full-length DNA sequence of Wuzhishan pig TLR 4 gene was 10 435 bp in length,of which CDS was 2 526 bp in length and encoded 841 amino acids.Compared with the common pig TLR 4 gene sequence published by GenBank,there were 38 mutations,of which two mutations were in the CDS region.The results of homology analysis showed that the amino acid sequence of the TLR 4 gene of Wuzhishan pig shared 99.9%,80.7%,79.9%,74.8%,74.7%,73.1%,73.0%,72.7%,62.5% and 43.4% homology with those of common pig,cattle,sheep,dog,horse,macaque,human,chimpanzee,mouse and chicken,respectively.The protein encoded by the TLR 4 of Wuzhishan pig belonged to secreted protein and transmembrane protein.It was hydrophilic,and had 8 glycosylation modification sites,17 serines (ser),7 threonines (Thr) and 8 tyrosine (TYR) phosphorylation sites.The amino acid phylogenetic tree analysis showed that the TLR 4 genes of different species are highly conserved during evolution.This result lays a foundation for further study of the function of TLR 4 gene.
基金Supported by Natural Science Foundation of Hainan Province(30515)~~
文摘[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.
文摘[ Objective] The study aimed to lay a foundation for studying immune recognition mechanism in xenotransplantation. [ Method] SLA- DQA gene of Hainan Wuzhishan miniature pig was cloned by RT-PCR and sequenced. J-Result] The SLA-DQA gene sequence of Wuzhishan minia- ture pig contained 768 nucleotides, among which 1 -765 were the complete open reading frame, encoding 255 amino acids. SLA-DQA cDNA se- quence and its deduced amino acid sequence of Wuzhishan miniature pig were most similar to those of the other miniature pigs in China. When compared with corresponding cDNA sequences and the amino acid sequences in other species, the homology with human was obviously higher than that with mouse. [ Conclusion] As the Chinese pig breed suitable for xenotransplantation, Wuzhishan miniature pig has significant importance to ser- ial studies on immunogenetics characteristics.
基金supported by the National High Technology Research and Development Program of China (2012AA020603)the National Transgenic Major Project, China (2008ZX08012-002-05)the National Key Technoligy R&D Program of China (2012BA13904)
文摘Skin grafting has been used as one of the most reliable tests to determine the genetic stability of laboratory animal such as mice and rats inbred line, but no identification of swine inbred lines by skin grafting has been reported. At present, Wuzhishan miniature pig （WZSP） inbred line has acquired the F24 individuals in China. In order to verify whether WZSP inbred line had D^en cultivated successfully, allogeneic skin grafts and related research were performed on F20 individuals of WZSP inbreeding population, compared with a control group of autologous transplantation. We observed the transplant recipients＇ wounds, detected peripheral blood-related indicators interleukin-2, 4 and 10, CD4~ and CD8~ lymphocytes, and conducted hematoxylin-eosin （HE） and Masson＇s staining of skin to judge whether the immune rejection reactions occurred within 28 days after transplantation. Chr. 7 genomic heterozygosity of 48 WZSP individuals from F20 to F22 was analyzed by high-density single nucleotide polymorphism （SNP） chips （60 000 SNPs）. The result showed that there were no significant differences in graft skin, the plasma interleukin-2, 4, 10, CD4~ and CD8~, HE and Masson＇s staining results between the allograft and autograft groups, and no immune rejection occurred on the allograft group. We found that 11 genes in Chr. 7 of major histocompatibility complex （MHC） I and MHC II were homozygous which confirmed that immune antibody of the allograft and autograft groups were highly identical and also provided a theoretical basis to no immune rejection occurred on the allograft in the inbred WZSP. The result proved that the WZSP inbred line had been cultivated successfully for the first time in the world. The test methods also provide a scientific basis for the identification of swine and mammal inbred lines.
