научная статья по теме MOLECULAR CHARACTERISTICS OF THE SLA-2 GENE FROM CHINESE HEBAO PIGS Биология

Текст научной статьи на тему «MOLECULAR CHARACTERISTICS OF THE SLA-2 GENE FROM CHINESE HEBAO PIGS»

ГЕНЕТИКА, 2012, том 48, № 2, с. 253-259

ГЕНЕТИКА ЖИВОТНЫХ =

УДК 575.113.1:599.731.1

MOLECULAR CHARACTERISTICS OF THE SLA-2 GENE FROM CHINESE HEBAO PIGS

© 2012 г. F.-Sh. Gao1, J. Bai1, X.-H. Zhang2, W.-J. Zhang2, D. Guo2, Sh. Zhang2

department of Biochemistry and Molecular Biology, College of Bioengineering, Dalian University,

Dalian, Liaoning 116622, China e-mail: gfsh0626@126.com 2Institute of Animal Husbandry of Liaoning Province, Liaoyang, Liaoning 111000, China Received May 16, 2011

To study the molecular characteristics of swine leukocyte antigen (SLA) class I from the Hebao pig, a rare inbreed in China, a pair of primers was designed to amplify the SLA-2 gene (SLA-2-HB) and then the molecular characteristics of the gene were analyzed by computer. After cloning, sequencing and computer analysis, four SLA-2-HB alleles were found, all of 1119 bp. Sites 3—1097 were an open reading frame encoding 364 amino acids with two sets of intra-chain disulfide bonds comprising four cysteines situated in sites 125, 188, 227 and 283. By alignment of SLA-2-HB sequences with other SLA-2 alleles in the DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank database, nine key variable amino acid sites were found in the extracellular domain of the SLA-2-HB alleles at sites 23(F), 24(I), 43(A), 44(K), 50(Q), 73(N), 95(I), 114(R) and 216(S), which could be used to differentiate other SLA-2 alleles. The amino acid identities between SLA-2-HB and other SLA-2, SLA-3 and SLA-1 alleles were 87.1-97.0%, 85.0-93.9% and 83.388.6%, respectively. The phylogenetic tree of SLA-2-HB showed that it was relatively independent of the other SLA-2 genes. Furthermore, the SLA-2-HB alleles were similar to HLA-B15 and HLA-A2 functional domains and preserved some functional sites of HLA-A2. It was concluded that SLA-2-HB is an allele of SLA-2 and that the Hebao pig might have evolved independently in China.

The Hebao pig has inbred for more than 300 years in enclosed mountainous terrain. Because of the harsh conditions of barren soil, dry weather and rough foraging, the Hebao pig displays many favorable characteristics such as stable genetics, earlier maturation, high fecundity, strong resistance against diseases and good weight gain on the roughest of forage. The meat of the Hebao pig is called 'Northern Sweet Pork' because of its spicy taste and pleasant aroma, though it is now a protected breed in China. Research has been undertaken on Hebao pig production, and until now there has been no study on its molecular characteristics.

The major histocompatibility complex (MHC) is a crucial cluster of immune response genes present in all vertebrate species [1]. In mammals, the MHC is divided into class I, II and III. MHC class I genes show more variation in their evolution than class II and III genes, which are relatively conserved [2]. The genomic structure, molecular characteristics and functions of MHC have been elucidated in humans, rats and other vertebrates.

Swine MHC, also termed swine leukocyte antigen (SLA), was discovered by Vaiman in 1970 [3]. The SLA cluster of genes is divided into three groups of linked genes: SLA class I (SLA-I), SLA class II (SLA-II) and

SLA class III (SLA-III). SLA-I has three functional loci: SLA-1, SLA-2 and SLA-3 [4, 5]. Among these, the SLA-2 locus is easily distinguished from SLA-1 and SLA-3 by substantial differences in their 5' and 3' untranslated regions. A further dissimilarity to the SLA-1 and SLA-3 loci is in three amino acid residues at the start of the signal peptide [6]. The SLA-2 locus might have a more crucial role as an SLA-I molecule (the roles of which include binding and presenting antigen molecules) because it is more polymorphic than the other two SLA-I loci [3, 5, 7].

Hebao pig is a unique breed reared in China. To study its molecular characteristics, a cloning scheme for Hebao pig SLA-2 was designed and its molecular evolution analyzed using molecular biology software.

MATERIALS AND METHODS

Breed, vector and bacterial strain. Hebao pigs were bred on a farm belonging to the Institute of Animal Husbandry of Liaoning Province in China. Fresh spleen tissues were removed from four pigs for analysis.

pMD18-T easy vector, Escherichia coli JM109, avian myeloblastosis virus (AMV) reverse transcriptase, isopropyl p-D-1-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl p-D-galacto-

The listed SLA-2 alleles from different breeds of swine

Accession number Breed

EU432083 From SK-RST cell line

AF464039 Hanford

EU432090 From ESK-4 cell line

EF125047 Large White

DQ104338 Sinclair

AB205147 Outbred, China

EU432087 From MPK cell line

EU170458 From PK15 cell line

AF464004 Yucatan

AF464023 NIH

DQ303223 Meishan

DQ187333 Korean native pig

DQ883210 Korean native pig

DQ992495 From KNP2004 clone

DQ992494 From KNP2003 clone

EU867814 Miniature NIH pig

AB231907 Bama miniature pig

AY247773 Westran

EU432088 From LLC-PK1 cell

AY135599 Hanford

AF464003 Yucatan

EF589960 Korean native pig

AF464005 Yucatan

AY247774 Large white

AF464058 Sinclair

DQ303222 Meishan

EU432089 From ESK-4 cell line

AF464049 Meishan

AF464059 Meishan

EU432085 From PT-K75 cell line

AY135598 Hanford

GQ261029 SNU miniature pig

DQ187334 Korean native pig

AY247775 Large White

DQ104339 Sinclair

EU431219 CMS miniature pig

EU432086 From MPK cell line

DQ187332 Korean native pig

DQ992500 From KNP2009 clone

DQ992497 From KNP2006 clone

DQ992498 From KNP2007 clone

DQ992499 From KNP2008 clone

DQ883211 Korean native pig

DQ992496 From KNP2005 clone

pyranoside (X-gal), T4 DNA Ligase and EcoRI restriction endonuclease were purchased from Takara Biotechnology. The TRIzol Total RNA Extraction Kit was purchased from Invitrogen Biotechnology. The GeneClean kit was purchased from BIO 101, Inc.

Primer design. To amplify the SLA-2 gene from He-bao pig, a pair of primers was designed with the reference to the reported SLA-2 (AF464049). The forward primer was S1 (5'-AGATGCGGGTCAGGGGCCCT-CAAG-3') and the reverse primer was S2 (5'-CAGTC-CCCACAAGGCAGCTGTCTC-3').

Amplification of SLA-2 by RT-PCR. Spleens were removed from four slaughtered Hebao pigs. One hundred milligrams of tissue was cut into pieces and placed in 1.5-mL Eppendorf tubes to which was added 300 |L TRIzol reagent. The pieces of tissue were homogenized using a pestle until a homogenate was formed. A further 700 |L of TRIzol reagent was added to the tube and the total mRNA extracted according to the kit instructions. The isolated total RNA was dissolved in 20 |L of 0.1% diethylpyrocarbonate (DEPC) and incubated for 5 min at 60°C, then stored at -80° for RT-PCR.

For reverse transcription, 2 |g of extracted total mRNA was mixed with 2 |L of Oligo (dT)15 (100 |mol/L), 2 |L ofAMV buffer, 4 |L of deoxynucle-otidey triphosphate (2.5 mmol/L) and 1 |L of AMV reverse transcriptase (5 U), at a final volume of 20 |L achieved by adding 0.1% DEPC water. The mixture was incubated at 42°C for 1 h to synthesize first-strand cDNA.

After reverse transcription, 2 |L (about 0.5 |g) of first-stand cDNA was used for PCR in a volume of 50 |L. The PCR reaction was as follows: 94°C for 5 min for pre-denaturation, then 94°C for 1 min, 65°C for 45 s and 72°C for 1 min for 30 cycles, the final extension at 72°C for 10 min. The PCR product was stored at -20°C for gene cloning.

Cloning of SLA-2. The PCR products were separated on a 1% agarose gel by 1x Tris-acetate-EDTA running buffer electrophoresis. The separated DNA was purified using a DNA recovery kit, and then the purified DNA was ligated to pMD 18-T easy vector according to the manufacturer's recommendations. The mixture was incubated at 4°C overnight, and then transformed into competent E. coli JM109 coated on LB plates containing ampicillin (100 |g/mL), IPTG (40 |g/mL) and X-gal (200 |g/mL). Following incubation at 37°C overnight, positive clones were screened by blue-white spots and restriction enzyme analysis using Eco RI and HindIII.

Gene sequencing and analysis. Nucleotide sequencing for each SLA-2 positive clone (at least 3 clones every one SLA-2 allele) was performed in both forward and reverse direction using an automated gene sequencing facility at Takara Bio Inc. In the light of sequencing results, the consensus sequences of the different clones for each SLA-2 allele from one animal was selected and submitted to the DNA Da-

tabank of Japan (DDBJ)/GenBank database through the SAKURA system. The GenBank accession numbers of the SLA-2 genes are listed in table. Alignments were performed using ClustalW with additional hand regulation; the deduced amino acid sequences were compared using the search similarity and multiple alignment programs of GENETYX version 9.0 computer software (Software Development Co., Ltd, Tokyo, Japan) and DNAMAN version 5.2.2 (Lynnon BioSoft Co., Ltd). The molecular phylogenetic tree was made using neighbor-joining method mapping in DNAMAN and Mega2 software (Mega Software Co., Ltd). The variance of the difference was computed using the bootstrap method (1000 replicates) and the substitutions type is amino acids along with p-distance model.

RESULTS

Cloning of the Hebao SLA-2 genes. PCR amplification of the four SLA-2 alleles resulted in 1119 bp fragments that were named SLA-2-HB01—04, covering an open reading frame (ORF) in sites 3—1097 encoding 364 amino acids. The first 24 amino acid residues constitute a signal peptide. Two sets of cysteines that are likely to form intra-chain disulfide bridges are present at sites 125, 188, 227 and 283. The SLA-2-HB alleles were submitted to the DDBJ/European Molecular Biology Laboratory (EMBL)/GenBank database and received accession numbers AB602431, AB602432, AB602433 and AB602434.

Alignment of SLA-2-HB and other SLA-2 alleles. By alignment of the SLA-2-HB sequences with other SLA-2 alleles in the DDBJ/EMBL/GenBank database, nine key variable amino acid sites were found in the extracellular domain of the SLA-2-HB alleles at sites 23(F),

Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.

Показать целиком