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).

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.