Production of a new tetravalent vaccine targeting fimbriae and enterotoxin of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is an important type of pathogenic bacteria that causes diarrhea in pigs. The objective of this study was to prepare a novel tetravalent vaccine to effectively prevent piglet diarrhea caused by E. coli. In order to realize the production of K88ac-K99-ST1-LTB tetravalent inactivated vaccine, the biological characteristics, stability, preservation conditions, and safety of the recombinant strain BL21(DE3) (pXKKSL4) were studied, and the vaccine efficacy and minimum immune dose were measured. The results indicated that the biological characteristics, target protein expression, and immunogenicity of the 1st to 10th generations of the strain were stable. Therefore, the basic seed generation was preliminarily set as the 1st to 10th generations. The results of the efficacy tests showed that the immune protection rate could reach 90% with 1 minimum lethal dose (MLD) virulent strain attack in mice. The immunogenicity was stable, and the minimum immune dose was 0.1 mL per mouse. Our research showed that the genetically engineered vaccine developed in this way could prevent piglet diarrhea caused by enterotoxigenic E. coli through adhesin and enterotoxin. In order to realize industrial production of the vaccine as soon as possible, we conducted immunological tests and production process research on the constructed K88ac-K99-ST1-LTB tetravalent inactivated vaccine. The results of this study provide scientific experimental data for the commercial production of vaccines and lay a solid foundation for their industrial production.

Escherichia coli entérotoxinogènes (ETEC) est un type important de bactéries pathogènes qui cause de la diarrhée chez les porcs. L'objectif de l'étude était de préparer un nouveau vaccin tétravalent pour prévenir efficacement la diarrhée causée par E. coli chez les porcelets. Afin de réaliser la production du vaccin tétravalent inactivé K88ac-K99-ST1-LTB, les caractéristiques biologiques, la stabilité, les conditions de conservation, et la sécurité de la souche recombinante (BL21(DE3)(pXKKSL4) ont été étudiées et l'efficacité du vaccin et la dose immunitaire minimum ont été mesurées. Les résultats indiquent que les caractéristiques biologiques, l'expression des protéines cibles, et l'immunogénicité de la 1ère à la 10e génération de la souche étaient stables. Ainsi, la génération germinale de base a été établie de manière préliminaire comme étant de la 1ère à la 10e générations. Les résultats des tests d'efficacité ont démontré que le taux de protection immunitaire pouvait atteindre 90 % avec une attaque au moyen de 1 dose léthale minimale (MLD) d'une souche virulente chez les souris. L'immunogénicité était stable et la dose immunitaire minimum était de 0,1 mL par souris. Nos travaux ont démontré que le vaccin génétiquement élaboré développé de cette façon pourrait prévenir la diarrhée chez les porcelets causée par des E. coli entérotoxigénique via les adhésines et les entérotoxines. Afin d'atteindre la production industrielle de ce vaccin aussitôt que possible, nous avons mené des tests immunologiques et de la recherche sur le processus de production du vaccin tétravalent inactivé K88ac-K99-ST1-LTB. Les résultats de la présente étude fournissent des données scientifiques expérimentales pour la production commerciale de vaccins et jettent une base solide pour leur production industrielle.(Traduit par Docteur Serge Messier).

Screening of linear B-cell epitopes and its proinflammatory activities of Haemophilus parasuis outer membrane protein P2.

Haemophilus parasuis is a commensal organism of the upper respiratory tract of pigs, but virulent strains can cause Glässer's disease, resulting in significant economic losses to the swine industry. OmpP2 is an outer membrane protein of this organism that shows considerable heterogeneity between virulent and non-virulent strains, with classification into genotypes I and II. It also acts as a dominant antigen and is involved in the inflammatory response. In this study, 32 monoclonal antibodies (mAbs) against recombinant OmpP2 (rOmpP2) of different genotypes were tested for reactivity to a panel of OmpP2 peptides. Nine linear B cell epitopes were screened, including five common genotype epitopes (Pt1a, Pt7/Pt7a, Pt9a, Pt17, and Pt19/Pt19a) and two groups of genotype-specific epitopes (Pt5 and Pt5-II, Pt11/Pt11a, and Pt11a-II). In addition, we used positive sera from mice and pigs to screen for five linear B-cell epitopes (Pt4, Pt14, Pt15, Pt21, and Pt22). After porcine alveolar macrophages (PAMs) were stimulated with overlapping OmpP2 peptides, we found that the epitope peptides Pt1 and Pt9, and the loop peptide Pt20 which was adjacent epitopes could all significantly upregulated the mRNA expression levels of IL-1α, IL-1ß, IL-6, IL-8, and TNF-α. Additionally, we identified epitope peptides Pt7, Pt11/Pt11a, Pt17, Pt19, and Pt21 and loop peptides Pt13 and Pt18 which adjacent epitopes could also upregulate the mRNA expression levels of most proinflammatory cytokines. This suggested that these peptides may be the virulence-related sites of the OmpP2 protein, with proinflammatory activity. Further study revealed differences in the mRNA expression levels of proinflammatory cytokines, including IL-1ß and IL-6, between genotype-specific epitopes, which may be responsible for pathogenic differences between different genotype strains. Here, we profiled a linear B-cell epitope map of the OmpP2 protein and preliminarily analyzed the proinflammatory activities and effects of these epitopes on bacterial virulence, providing a reliable theoretical basis for establishing a method to distinguish strain pathogenicity and to screen candidate peptides for subunit vaccines.

Preparation of novel trivalent vaccine against enterotoxigenic Escherichia coli for preventing newborn piglet diarrhea.

OBJECTIVE: To develop a trivalent genetically engineered inactivated Escherichia coli vaccine (K88ac-3STa-LTB) that neutralizes the STa toxin by targeting fimbriae and entertoxins for the treatment of enterotoxigenic E coli. ANIMALS: 18- to 22-g mice, rabbits, pregnant sows. PROCEDURES: Using PCR, the K88ac gene and LTB gene were cloned separately from the template C83902 plasmid. At the same time, the 3 STa mutant genes were also amplified by using the gene-directed mutation technology. Immune protection experiments were performed, and the minimum immune dose was determined in mice and pregnant sows. RESULTS: The ELISA test could be recognized by the STa, LTB, and K88ac antibodies. Intragastric administration in the suckling mouse confirmed that the protein had lost the toxicity of the natural STa enterotoxin. The results of the immune experiments showed that K88ac-3STa-LTB protein could stimulate rabbits to produce serum antibodies and neutralize the toxicity of natural STa enterotoxin. The efficacy test of the K88ac-3STa-LTB-inactivated vaccine showed that the immune protection rate of the newborn piglets could reach 85% on the first day after suckling. At the same time, it was determined that the minimum immunization doses for mice and pregnant sows were 0.2 and 2.5 mL, respectively. CLINICAL RELEVANCE: This research indicates that the K88ac-3STa-LTB trivalent genetically engineered inactivated vaccine provides a broad immune spectrum for E coli diarrhea in newborn piglets and prepares a new genetically engineered vaccine candidate strain for prevention of E coli diarrhea in piglets.

Development of a new candidate vaccine against piglet diarrhea caused by Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is an important type of pathogenic bacteria that causes diarrhea in humans and young livestock. The pathogen has a high morbidity and mortality rate, resulting in significant economic losses in the pig industry. To effectively prevent piglet diarrhea, we developed a new tetravalent genetically engineered vaccine that specifically targets ETEC. To eliminate the natural toxin activity of ST1 enterotoxin and enhance the preventive effect of the vaccine, the mutated ST 1, K88ac, K99, and LT B genes were amplified by PCR and site-specific mutation techniques. The recombinant strain BL21(DE3)(pXKK3SL) was constructed and achieved high expression. Animal experiments showed that the inactivated vaccine had eliminated the natural toxin activity of ST1. The immune protection test demonstrated that the inclusion body and inactivated vaccine exhibited a positive immune effect. The protection rates of the inclusion body group and inactivated vaccine group were 96 and 98%, respectively, when challenged with 1 minimum lethal dose, indicating that the constructed K88ac-K99-3ST1-LTB vaccine achieved a strong immune effect. Additionally, the minimum immune doses for mice and pregnant sows were determined to be 0.2 and 2 mL, respectively. This study suggests that the novel K88ac-K99-3ST1-LTB vaccine has a wide immune spectrum and can prevent diarrhea caused by ETEC through enterotoxin and fimbrial pathways. The aforementioned research demonstrates that the K88ac-K99-3ST1-LTB vaccine offers a new genetically engineered vaccine that shows potential for preventing diarrhea in newborn piglets.

NPC1L1 Plays a Novel Role in Nonalcoholic Fatty Liver Disease.

Niemann-Pick C1-Like 1 (NPC1L1) is a key protein in the transport of cholesterol, which exists in the brush marginal membrane of the intestinal epithelial cells and the timid duct membrane of the liver. It affects cholesterol absorption and plasma low-density lipoprotein levels. Cholesterol is both an important component of the cell membrane and a precursor of bile acid and steroid hormone synthesis. Abnormal cholesterol metabolism is closely related to nonalcoholic steatohepatitis (NASH). NASH can progress to fibrosis and cirrhosis, with serious consequences. NPC1L1 is involved in the regulation of cholesterol and lipid metabolism and plays an important role in maintaining the balance of cholesterol metabolism in the body. It also plays an important role in some metabolic diseases such as nonalcoholic fatty liver disease, obesity, and hypercholesterolemia. Therefore, it is necessary to elucidate the molecular pathological mechanism of NPC1L1 in the regulation of cholesterol metabolism and the occurrence and development of NASH, which can provide a target for the development of novel drugs for the treatment of NASH and other diseases. More importantly, it helps to accelerate the development of drugs that regulate lipid metabolism at multiple levels and reduce liver steatosis, which is extremely important for the prevention and treatment of NASH and related severe metabolic diseases.

Molecular structure and function of the carboxy-terminus of the alpha-toxin from Clostridium perfringens type A.

In order to interpret the molecular structure and biological characteristics of Clostridium perfringens alpha-toxin (CPA), the CPA251-370 gene was cloned and the 120 amino acid carboxy terminal of CPA (CPA251-370) was obtained. The secondary and three-dimensional (3D) structures of CPA251-370 were predicted. The secondary structure of CPA251-370 consisted primarily of 35.48% ß-sheets and 44.35% random coils. Compared with the CPA toxin consisting of 10 α-helices and eight ß-sheets, the 3D structure of CPA251-370 only contained eight ß-sheets. The circular dichroism (CD) spectrum detection showed that the CD spectrum of CPA251-370 changed slightly compared with the CD spectrum of CPA. Biological activity assays showed that CPA251-370 had lost the phospholipase C (PLC) activity and haemolytic activity of CPA. More importantly, the mice immunized with CPA251-370 were protected against a challenge with 1 MLD C. perfringens type A strain C57-1. This study laid a solid foundation for explaining the relationship between molecular structure and biological characteristics of CPA in the future. Our research also provides CPA251-370 as a candidate strains for genetic engineering subunit vaccines of C. perfringens type A.

Study of the Structure and Biological Activity of the Amino-Terminus of the α-Toxin from Clostridium welchii Type A.

To explore the biological activity of Clostridium welchii α-toxin (CPA), the Asp56 residue of CPA was mutated to glycine (CPA D56G) by site-directed mutagenesis, and the 250 amino acid amino-terminal phospholipase C (PLC)-containing domain of CPA (PLC1-250) was isolated. The secondary and three-dimensional (3D) structures of CPA D56G and PLC1-250 were predicted, and the results showed that the secondary structures of CPA D56G and PLC1-250 were composed of α-helices and random coils. The 3D structures of CPA D56G and PLC1-250 were similar to the 3D structures of CPA. The circular dichroism (CD) spectrum of CPA D56G differed from the CD spectrum of CPA, but the CD spectrum of PLC1-250 was similar to the CD spectrum of CPA. Biological activity assays showed that CPA D56G lost the PLC activity of CPA and that mice immunized with CPA D56G were protected against a challenge with 1 MLD C. welchii type A strain C57-1. In addition, PLC1-250 contained the PLC activity of CPA. This study laid a solid foundation for future studies on the relationship between the molecular structure and biological function of CPA and its molecular mechanism. Our study also provided CPA D56G as a candidate strain for engineering a CPA subunit vaccine for C. welchii type A.

CDC42-Interacting Protein 4 Gene Is Down Trans-Regulated by HBV DNA polymerase Trans Activated Protein 1.

BACKGROUND: Hepatitis B Virus (HBV) DNA polymerase transactivated protein 1 (HBVDNAPTP1) is a novel protein transactivated by HBV DNA polymerase, screened by suppression subtractive hybridization technique (GenBank accession no: AY450389). The biological function of HBVDNAPTP1 was investigated in this study. METHODS: We constructed a vector pcDNA3.1 (-)/myc-His A-HBVDNAPTP1 and used it to transfect acute monocytic leukemia cell line THP-1. HBVDNAPTP1 expression was detected by western blot analysis in the cells. A cDNA library of genes transactivated by HBVDNAPTP1 in THP-1 cells was made in pGEM-T Easy using suppression subtractive hybridization (SSH). The cDNAs were sequenced and analyzed with BLAST search against the sequences in GenBank. RESULTS: Some sequences, such as CIP4, might be involved in apoptosis development. mRNA and protein expression of CIP4 was identified by Real time RT-PCR and western blot in THP-1 cells. HBVDNAPTP1 could down-regulate the expression of CIP4 at both transcription and translation levels. CONCLUSION: HBVDNAPTP1 may be involved in the positive regulation on the initiation of monocyte apoptosis. The result contribute to reveal the HBVDNAPTP1 biological functions and provide new evidences for further exploration of the regulatory mechanism of HBVDNAPTP1.

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.

[Analyzing secondary structure of beta2 microglobulin in Escherichia coli with circular dichroism spectrum].

OBJECTIVE: In order to study the structure and function of beta2 microglobulin (beta2 m). METHODS: We sub-cloned the mature peptide of beta2 m into the p2X plasmid and transformed them to Escherichia coli TB1. The recombinant bacterium was induced to be expressed and the expressed fusion protein was detected by SDS-PAGE and western blot. After purifying and cleaving with Factor Xa, we separated the monomer protein beta2 m from MBP. Finally, we determined the secondary structure of the beta2 m protein by circular dichroism (CD) spectrum. RESULTS: MBP-beta2 m was 52.1 kDa, and the monomer protein beta2 m was 10.6 kDa. The alpha-helix, beta-sheet, turn, and random coil of the fusion protein showed 0, 45, 8 and 45 amino acids, respectively, detected by CD estimation. CONCLUSION: The beta2 m protein had correct secondary structure and could be used for further research of peptide binding in vitro.

[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.