Construction of multiple recombinant SLA-I proteins by linking heavy chains and light chains in vitro and analyzing their secondary and 3-dimensional structures.

Six breeds of swine were used to study the structure of swine leukocyte antigen class I (SLA-I). SLA-I complexes were produced by linking SLA-2 genes and ß(2)m genes via a linker encoding a 15 amino acid glycine-rich sequence, (G4S)3, using splicing overlap extension (SOE)-PCR in vitro. The six recombinant SLA-2-linker-ß(2)m genes were each inserted into p2X vectors and their expression induced in Escherichia coli TB1. The expressed proteins were detected by SDS-PAGE and western blotting. The maltose binding protein (MBP)-SLA-I fusion proteins were purified by amylose affinity chromatography followed by cleavage with factor Xa and separation of the SLA-I protein monomers from the MBP using a DEAE Ceramic Hyper D F column. The purified SLA-I monomers were detected by circular dichroism (CD) spectroscopy and the 3-dimensional (3D) structure of the constructed single-chain SLA-I molecules were analyzed by homology modeling. Recombinant SLA-2-Linker-ß(2)m was successfully amplified from all six breeds of swine by SOE-PCR and expressed as fusion proteins of 84.1 kDa in pMAL-p2X, followed by confirmation by western blotting. After purification and cleavage of the MBP-SLA-I fusion proteins, SLA-I monomeric proteins of 41.6 kDa were separated. CD spectroscopy demonstrated that the SLA-I monomers had an α-helical structure, and the average α-helix, ß-sheet, turn and random coil contents were 21.6%, 37.9%, 15.0% and 25.5%, respectively. Homology modeling of recombinant single-chain SLA-I molecules showed that the heavy chain and light chain constituted SLA-I complex with an open antigenic peptide-binding groove. It was concluded that the expressed SLA-I proteins in pMAL-p2X folded correctly and could be used to bind and screen nonameric peptides in vitro.

Secondary structure and 3D homology modeling of swine leukocyte antigen class 2 (SLA-2) molecules.

No information to date is available to elucidate the structure of swine leukocyte antigen class I (SLA-I) molecule which is comprised by a heavy chain of SLA-I non-covalently associated with a light chain, beta(2)-microglobulin (beta(2)m) proteins. Presently, one of SLA-I gene SLA-2 and beta(2)m gene were expressed as soluble maltose binding proteins (MBP-proteins) in a pMAL-p2X/Escherichia coli TB1 system and identified by western blotting with anti-MBP polyclonal antibodies. The expressed proteins MBP-SLA-2 and MBP-beta(2)m were purified on amylose affinity columns followed by DEAE-Sepharose. The purified products were cleaved by Factor Xa, respectively, and the interest of proteins SLA-2 and beta(2)m were purified on amylose affinity columns followed by separation from MBP on DEAE-Sepharose. The secondary structures of SLA-2 and beta(2)m were analyzed by circular dichroism (CD) spectrophotometry. The three-dimensional (3D) structure of their peptide-binding domain (PBD) was modeled-based sequence homology. The content of the alpha-helix, beta-sheet, turn, and random coil in the SLA-2 protein were 76, 95, 36, and 67aa, respectively. In the 98aa of beta(2)m, the contents of the alpha-helix, beta-sheet, turn, and random coil were 0, 45, 8, and 45aa, respectively. The SLA-2 protein displayed a typical alpha-helix structure while beta(2)m protein displayed a typical beta-sheet structure. Homology modeling of the SLA-2 and beta(2)m proteins demonstrated similarities with the structure of human and mouse MHC (major histocompatibility complex) class I proteins.

[Expression of alpha-toxin gene of Clostridium perfringens type A and its primary immunological protective function].

Alpha-toxin gene was amplified from chromosomal DNA of Clostridium perfringens type A by polymerase chain reaction (PCR). PCR product was inserted into vector pGEM-T directly. The cloned recombinant plasmid pXCPA02 possesses positive nucleotide sequence of alpha-toxin. A 1.2 kb alpha-toxin gene fragment was cleaved with restriction endonucleases Nco I /EcoR I from plasmid pXCPA02, and then inserted into an expression vector pET-28c which cleaved with Nco I /EcoR I by blunt-end ligation. The recombinant expression plasmid pXETA02 was studied in detail by restriction endonucleases analysis and nucleotide sequencing. The results showed that the recombinant expression pXETA02 possessed a positive alpha-toxin gene sequence and reading frame. BL21 (DE3) (pXETA02) could produce alpha-toxin and the expressed products were recognized by alpha-toxin monoclonal antibodies with ELISA and Western blot. The expression optimization result indicated that the alpha toxin gene expression optimized condition with IPTG induction is culture medium pH 7.5, culture temperature 37 degrees C, joining IPTG to final concentration 0.8 mmol/L when the recombinant strain growth density OD600 achieved 0.8, and induction time 5h. The expression level of the alpha-toxin proteins were about 34.28% of total cellular protein with IPTG induction by SDS-PAGE and thin-layer gel scanning analysis. The alpha toxin gene expression optimized condition with lactose induction is culture medium pH 7.5, culture temperature 37 degrees C, joining lactose to final concentration 0.1 g/L when the recombinant strain growth density OD600 achieved 0.8, and induction time 5h. The expression level of the alpha-toxin proteins were about 23.82% of total cellular protein with lactose induction by SDS-PAGE and thin-layer gel scanning analysis. More importantly, Immunization in a mouse model with crude preparation containing the alpha-toxin protein inclusion bodies or inactivated recombinant strain induced protection against at least 1 MLD of the toxin from Clostridium perfringens type A.

[Fusion of betal-toxin gene and beta2-toxin gene from Clostridium perfringens type C].

Betal-toxin and beta2-toxin genes from chromosomal DNA of Clostridium perfringens type C were amplified by PCR, PCR products were cleaved with restriction endonucleases and recovered. The recombinant plasmid pETXB1-2 containing beta1-beta2 fusion genes was constructed by recombinant technique and then transformed into Escherichia coli BL21 (DE3). The beta1-beta2 fusion proteins were expressed in recombinant strain BL21 (DE3) (pETXB1-2), and the expression level of the beta1-beta2 fusion proteins was about 15.36% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis. More importantly, immunization in a mouse model with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain induced protection against at least 1 MLD of the toxin from Clostridium perfringens type C. Hence the fusion proteins possess a good immunogenicity. The constructed recombinant strain BL21 (DE3) (pETXB1-2) can be used as a candidate of vaccine strain.

[Construction of recombinant strain expressing enterotoxigenic Escherichia coli K88ac-ST1-LTB fusion protein].

K88ac genes, heat-stable enterotoxin I (ST1) mutant genes and heat-labile enterotoxin B subunit (LTB) genes from plasmids of Escherichia coli C83902 were amplified by PCR. The recombinant expression plasmid pXKST3LT5 containing K88ac-ST1-LTB fusion gene was constructed by recombinant DNA technique and then transformed into Escherichia coli BL21(DE3). The K88ac-ST1-LTB fusion protein was highly expressed in recombinant strain BL21 (DE3)(pXKST3LT5) and the expression level of the K88ac-ST1-LTB fusion protein was about 75.53% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis. More importantly, mice immunized with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain produced antibodies that were able to recognize ST1 in vitro. These sera antibodies were able to neutralize the biological activity of native ST1 in the suckling mouse assay. Hence the fusion protein was nontoxic and immunogenic, the constructed recombinant strain could be used as a candidate of vaccine strain.