Cloning α and β chains of SLA-DR loci and reconstruction of their complex

Expand
  • 1.Bioengineering College, Dalian University; College of Veterinary Medicine, China Agricultural University; 2.College of Veterinary Medicine, China Agricultural University; 3.The Fire Engineering Department, the Chinese People's Armed Police Forces Academy;

Published date: 05 Sep 2008

Abstract

In order to investigate the prompt conformations of swine major histocompatibility complex (MHC), a swine MHC-II protein complex (SLA-II) was reconstructed in vitro. DRA and DRB were cloned from a crossbred commercial pig (Changbai-Dalan). Subcloned extracellular parts of DRA and DRB were linked together by a linker containing rich glycine/serine (G4S)3, and the whole length of two genes, named DRA-linker-DRB, was amplified by splicing overlap extension PCR (SOE PCR). DRA-linker-DRB was inserted into pMAL-p2X prokaryotic system and expressed. The expressed fusion protein MBP-DRA-(G4S)3-DRB was identified by Western-blot, purified and cleaved to obtain the protein of interest DRA-(G4S)3-DRB. The secondary structure of this protein was determined in circular dichroism (CD) apparatus. The results indicated that the subsequent protein MBP-DRA-(G4S)3-DRB was soluble and its molecule weight was 83.4 ku in consistent with the Western-blot. Cleaved by Factor Xa, the protein of interest was separated with a molecular weight of 40.9 ku. The CD spectrum demonstrated that the protein displayed a favorable ?-Helix structure, and the contents of ?-Helix, ?-sheet, turn, and random coil were 80 aa, 121 aa, 101 aa and 80 aa respectively. The identical ratios of ?-Helix, ?-sheet, turn, and random coil between MBP-DRA-(G4S)3-DRB and DRA-(G4S)3-DRB were 95%, 96.7%, 91.1% and 93.0%, respectively. The results also suggested that the reconstructed SLA-II complex presented an ideal conformation and can be used for studying its structure and function in vitro.

Cite this article

GAO Fengshan, WANG Lei, LI Xinsheng, LI Yungang, FANG Qinmei, HAO Huifang, XIA Chun, WANG Huifei . Cloning α and β chains of SLA-DR loci and reconstruction of their complex[J]. Frontiers of Agriculture in China, 2008 , 2(3) : 355 -360 . DOI: 10.1007/s11703-008-0044-0

References

1. Bradford M N (1976). A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem, 72: 248–254. doi:10.1016/0003‐2697(76)90527‐3
2. Chen Y H, Yang J T, Martinez H M (1972). Determination of the secondary structuresof proteins by circular dichroism and optical rotary dispersion. Biochemistry, 11(22): 4120–4131. doi:10.1021/bi00772a015
3. Denkberg G, Cohen C J, Segal D, Kirkin A F, Reiter Y (2000). Recombinant humansingle-chain MHC-peptide complexes made from E. coli by in vitro refolding: functional single-chain MHC-peptide complexes and tetramerswith tumor associated antigens. Eur J Immunol, 30(12): 3522–3532. doi:10.1002/1521‐4141(2000012)30:12<3522::AID‐IMMU3522>3.0.CO;2‐D
4. Kapust R B, Waugh D S (1999). Escherichia coli maltose-binding proteinis uncommonly effective at promoting the solubility of polypeptidesto which it is fused. Protein Sci, 8(8): 1668–1674
5. Patrick M R, Buchbinder D, Jolley X, Dennett Buchanan R (1999). Incidence of inflammatory myopathies in Victoria,Australia, and evidence of spatial clustering. J Rheumatol, 26(5): 1094–1100
6. Srinivasan M, Domanico S Z, Kaumaya P T, Pierce S K (1993). Peptides of 23 residues or greater are required to stimulate a highaffinity class II-restricted T cell response. Eur J Immunol, 23(5): 1011–1016. doi:10.1002/eji.1830230504
7. Uger R A, Barber B H (1998). CreatingCTL targets with epitope-linked beta 2-microglobulin constructs. J Immunol, 160(4): 1598–1605
8. Wang J Y, Zhu S G, Xu C F (2002). Biochemistry. 3rd ed.Beijing: Higher Education Press, 197–252 (in Chinese)
9. Wang Y, Rubtsov A, Heiser R, White J, Crawford F, Marrack P, Kappler J W (2005). Usinga baculovirus display library to identify MHC class I mimotopes. Proc Natl Acad Sci USA, 102(7): 2476–2481. doi:10.1073/pnas.0409798102
10. White J, Crawford F, Fremont D, Marrack P, Kappler J (1999). Solubleclass I MHC with beta2-microglobulin covalently linked peptides: specificbinding to a T cell hybridoma. J Immunol, 162(5): 2671–2676
11. Xia C, Wu D, Wu W X, Wan J Q, Wang L, Yang T Y, Wang Q, Ning Y B (2005). Cloning and expression of interferon-alpha/gammafrom a domestic porcine breed and its effect on classical swine fevervirus. Vet Immunol Immunopathol, 104(1–2): 81–89. doi:10.1016/j.vetimm.2004.10.005
12. Yan R Q, Li X S, Yang T Y, Xia C (2005). Characterization of BF2 and beta(2)m in three Chinese chicken lines. Vet Immunol Immunopathol,108(3–4): 417–425. doi:10.1016/j.vetimm.2005.06.002
13. Yang D F, Zhu H F, Wang Z H, Shen G X, Tian D Y (2005). Construction of singlechain Fv antibody against transferrin receptor and its protein fusionwith alkaline phosphatase. World J Gastroenterol, 11(21): 3300–3303
Outlines

/