53results about How to "Comply with biosafety requirements" patented technology

The invention relates to a mouse-typhus salmonella gene-deletion mutant strain without containing resistance marks, a vaccine and application thereof, belonging to the technical field of animal bacterium genetic engineering. The invention utilizes a mediated allelic exchange technique of recombinant suicidal plasmid for obtaining the mouse-typhus salmonella gene-deletion mutant strain (Salmonellatyphimurium SL13441) (the preservation number is CCTCC NO: M2010301) without containing the resistance marks. The mutant strain is deficient in an important toxicity regulating gene-cya gene in mouse-typhus salmonella, so that the toxicity of the mouse-typhus salmonella is obviously weakened after the deficiency of the cya gene, but the mouse-typhus salmonella still has immunogenicity. The invention also discloses the Salmonella gullinarum gene-deletion vaccine prepared by utilizing the gene-deletion mutant strain and an application of the gene-deletion mutant strain in preparing the vaccine, and the prepared vaccine meets the requirement on biological safety. The gene-deletion vaccine prepared in the invention can effectively prevent infection of Salmonella gullinarum and has the advantages of good stability and safety and low cost, is convenient to use and has obvious economic and social benefits.

The invention belongs to the animal bacteria genetic engineering field, and relates particularly to construction of a vaccine for a serotype-7 double gene deletion mutation strain for differentiating actinobacillus pleuropneumoniae of swine of a vaccine immunized animal and a wild virus infected animal, and the application of the strain. In the invention, a serotype-7 double gene deletion mutation strain APP-7-mut01 for differentiating actinobacillus pleuropneumoniae of swine of a vaccine immunized animal and a wild virus infected animal (with accession number of CCTCC NO:M207004) is obtained. The strain deletes two main virulence factors of apxIIC and apxIVAN of APP serotype-7. The mutant strain is safe for swines, and has toxins of ApxIIA and ApxIVAC protein, thereby ensuring the immunity efficacy of the vaccine. The vaccine of the mutant strain is used to immunize animals, so that antibodies aiming at Apx IVAN protein are no longer produced inside the animals, however wild virus infection can produce antibodies aiming at ApxIVAN. The invention further discloses the construction, the vaccine preparation and the application of the double gene deletion mutation strain.

The invention belongs to the technical field of bacterial gene engineering of animals, and in particular relates to the construction of a recombinant salmonella choleraesuis strain which does not contain resistance markers and expresses major antigenic loci of porcine transmissible gastroenteritis virus, vaccine preparation and application. In the recombinant salmonella choleraesuis strain C500 (pYA-2SLN) which does not contain the resistance markers and expresses the major antigenic loci of the porcine transmissible gastroenteritis virus, the preservation No. is CCTCC NO: M 209189, and the strain misses asd genes which are necessary for the growth of salmonella choleraesuis and contains plasmid capable of expressing the asd genes, genes of an antigenic locus A, an antigenic locus D and an antigenic locus N321 of the porcine transmissible gastroenteritis virus in the strain. The invention also discloses a method for preparing the salmonella choleraesuis and porcine transmissible gastroenteritis vaccine by utilizing the recombinant strain and application thereof. The prepared recombinant vaccine can stimulate pigs to generate protective immune response for resisting the salmonella choleraesuis and the porcine transmissible gastroenteritis virus, and prevent the infection of the salmonella choleraesuis and the porcine transmissible gastroenteritis virus effectively.

The invention belongs to the technical field of vaccine preparation of animal gene engineering and particularly relates to the construction of a Bordetella bronchiseptica aroA (5-enolpyruvyl-shikimate-3-phosphate synthase) gene deleted bacterial strain, and the preparation and application of a gene deleted vaccine. The Bordetella bronchiseptica gene deleted bacterial strain QH0814DeltaaroA is preserved in the China Center for Type Culture Collection with the collection number of CCTCCNO: M208018. The deletion of 5-enolpyruvyl-shikimate-3-phosphate synthase (aroA) gene with the full length of 1340bp causes an obstacle when the bacterial strain metabolizes aromatic amino acid. The invention prepares the Bordetella bronchiseptica gene deleted vaccine by using the gene deleted bacterial strain. The invention also discloses the application of the gene deleted bacterial strain in preparing the Bordetella bronchiseptica gene deleted vaccine (attenuated live vaccine).

The invention discloses the construction of a recombinant Bordetella bronchiseptica strain which does not contain a resistance marker and is used for expressing an ORF2 gene fragment of porcine circovirus type 2, the preparation of a vaccine and application, and belongs to the field of genetic engineering. The recombinant Bordetella bronchiseptica strain QH0814delta aroA/ORF2 which does not contain the resistance marker and is used for expressing the ORF2 gene fragment of the porcine circovirus type 2 is obtained. In the strain, a 5-enolpyruvyl-shikimate-3-phosphate synthase (aroA) gene of Bordetella bronchiseptica is deleted; and the strain contains the ORF2 gene fragment of the porcine circovirus type 2. The invention discloses the preparation of the recombinant strain and the vaccine and application of the recombinant strain and the vaccine to the preparation of porcine circovirus type 2 and porcine Bordetella bronchiseptica vaccines. The genetic engineering vaccine can stimulate pigs to produce the protective immune reaction of resisting the porcine circovirus type 2 and porcine Bordetella bronchiseptica wild strains, and can effectively prevent the porcine circovirus type 2 and porcine Bordetella bronchiseptica from infecting the pigs.

The invention relates to a gene recombined swine cholera salmonella choleraesuis vaccine for a blue-ear disease and application thereof, belongs to the technical field of animal bacteria genetic engineering, and particularly relates to construction of recombined swine cholera salmonella choloraesuis without resistance maker and used for expressing main immunogenicity membrane protein of porcine reproductive and respiratory syndrome virus(PRRSV) and preparation and application of a live vaccine. Recombined swine cholera salmonella choleraesuis C501-GP5m expressing the swine PRRSV GP5m protein is gained and preserved in the China model culture preservation centre, and the preservation number is CCTCC NO: M2012275. The invention further discloses a method for preparing swine cholera salmonella choleraesuis and the porcine reproductive and respiratory syndrome virus vaccine from the recombination strain and application thereof. The prepared recombined live vaccine can stimulate swine to develop the immunity to resist swine cholera salmonella choloraesuis and PRRSV main immunogenicity membrane protein GP5m, and can effectively prevent the infection of paratyphus suum and PRRSV.

The invention discloses recombinant S. Pullorum, as well as a preparation method and an application thereof, belonging to the technical field of animal bacterial gene engineering. The name of the recombinant S. Pullorum is S. Pullorum C79-13deltacrpdeltaasd(pYA3493-HN), the preservation number is CCTCC NO:M 2015305, the newcastle diseases virus protective antigen gene-HN gene expressed by the recombinant S. Pullorum is the important immunogenic gene segment for the newcastle diseases virus, and has good immune protection. The recombination strain derives from the S. Pullorum standard strain C79-13, completely reserves the immune efficacy of C79-13 for S. Pullorum, has weaker toxicity than that of C79-13, has good bio-safety, and can simultaneously provide protection aiming at S. Pullorum and newcastle disease pathogenes. Meanwhile, the recombinant strain contains no resistance markers, and completely accords with the requirements on the bio-safety of vaccines.

The invention discloses a porcine actinobacillus pleuropeumoniae ClpP protease gene-deleted strain containing no resistance marker, and a construction method thereof. The invention belongs to the technical field of bacteria genetic engineering. According to the invention, a recombinant strain APPdeltaclpP of actinobacillus pleuropeumoniae is a strain obtained through coding gene deactivation upon ClpP protease in actinobacillus pleuropeumoniae with an oriented homologous recombination technology, and the expression of the ClpP protease is damaged. With the technology provided by the invention, the virulence of the mutant strain is lower than a parent strain, and the mutant strain is safe to animals. Therefore, an important basis is provided for the modification upon porcine contagious pleuropneumonia vaccines and the development of corresponding differential diagnosis reagents. The strain and the method provided by the invention have important significances in promoting the eradication and purification of porcine contagious pleuropneumonia all around the world.

The invention discloses a preparation method and application of attenuated live vaccine chitosan microsphere for a porcine epidemic diarrhea virus. The method comprises the following steps: culturinga porcine epidemic diarrhea virus attenuated strain PEDV-YN144, and filtering, so as to obtain virus filtrate; adding a sodium hydrogen carbonate solution into the virus filtrate, slowly dropwide adding a chitosan solution under a magnetic stirring condition; slowly dropwise adding a sodium tripolyphosphate solution; and stirring, so as to obtain mixed liquid; uniformly mixing gelatin with the mixed liquid, and carrying out freeze drying, so as to obtain the attenuated live vaccine chitosan microsphere for the porcine epidemic diarrhea virus. An organic reagent is not added in a packaging process adopted in the method, the antigen activity is preserved, the preparation process is simple, the time is saved, and the attenuated live vaccine chitosan microsphere is easy to store and transport.According to the attenuated live vaccine chitosan microsphere for the porcine epidemic diarrhea virus, the immune effect of a vaccine can be improved, and the action time of the vaccine can be prolonged.

The invention belongs to the technical field of animal bacteriosis gene engineering vaccines, particularly relates to strain construction, vaccine preparation and application of a recombined haemophilus parasuis attenuated salmonella vaccine strain without resistance makers and used for expressing a surface antigen hps 06257 of haemophilus parasuis, and aims to obtain a recombinant salmonella choleraesuis asd-C500/pYA-06257 (the preservation number is CCTCC NO: M2013054) without resistance makers and used for expressing the surface antigen HPS 06257 of haemophilus parasuis. The recombinant bacteria is lack of an asd gene on a salmonella choleraesuis genome, and contains a recombinant plasmid pYA-06257 which can express the asd gene and an outer membrane antigen hps 06257 gene of haemophilus parasuis on the strain. The invention further discloses a construction method of the recombinant strain asd-C500/pYA-06257 and a corresponding preparation method of the haemophilus parasuis attenuated salmonella vaccine, as well as application to the preparation of the haemophilus parasuis attenuated salmonella vaccine.

The invention discloses a resistance marker-free porcine actinobacillus pleuropneumoniae (APP) serum type 7 double-gene defective strain, and belongs to the technical field of bacterial gene engineering. The recombinant strain APPdeltaclpPdeltaapx II C of APP is obtained by inactivating ClpP protease in the APP and a coded gene of a hemolysin activated factor Apx II C by adopting a directional homologous recombination technology, and expression of the ClpP protease and the Apx II C protein is destroyed. The obtained double-gene defective strain has lower toxicity compared with a parent strain, is safe to animals, provides an important basis for transformation of porcine contagious pleuropneumonia (PCP) vaccines and research of matched identification and diagnosis reagents, and has great significance for promoting elimination and purification of the global PCP.

The invention relates to a salmonella choleraesuis gene deletion mutant C7821 without a resistant marker, with collection number being CCTCCNO: M2010102. The invention also relates to a salmonella choleraesuis gene deletion vaccine prepared by the mutant. The gene engineering strain of the mutant is derived from the salmonella choleraesuis C78-2 strain (which is a commercial strain purchased from the China Veterinary Culture Collection Center of China Institute of Veterinary Drugs Control). The main virulence adjusting gene crp of the mutant deletes 320bp, so that the bacteria can not ingest cAMP from the mammals and grow more slowly, and the virulence of the bacteria is greatly reduced, thus the mutant has high safety. The animal tests prove that the gene deletion mutant strain has good protective immunity. The gene deletion vaccine prepared by the invention can stimulate the swine to generate protective immune response resistant to salmonella choleraesuis homologous wild strains and effectively prevent infection with salmonella choleraesuis.

The invention discloses an engineering yeast strain for preventing a porcine circovirus disease. A gene engineering bacteria comprise genes for coding LSO fusion protein; the LSO fusion protein has a sequence shown as SEQ ID NO:1. A high-copying yeast integrating type plasmid surface is used for showing the fusion protein of a multiple-epitope antigen of the porcine circovirus PCV2 and a protective antigen LTB SLT IIeB of pathogenic swine colibacillosis; meanwhile, the recombinant fusion protein LTB SLT IIeB per se has the effect of an immunologic adjuvant, is used as both the antigen and an adjuvant, and achieves the dual functions; the immunogenicity and the immune efficiency of CAP protein coded by ORF2 of the porcine circovirus PCV2 shown by recombinant beer yeast strains are improved. The genetic engineering yeast strain can be applied to the prevention and treatment of the porcine circovirus disease.

The invention belongs to the technical field of gene engineering, and particularly relates to a recombinant adeno-associated virus transfer vector containing a gD protein gene of a variant porcine pseudorabies virus, a recombinant adeno-associated virus, and preparation and application thereof. The recombinant adeno-associated virus (rAAV) is used as a carrier to express the porcine pseudorabies virus gD protein, and the rAAV has the advantages of safety, effectiveness, specificity and the like. The genetically engineered strain prepared by the method expresses the gD immunogenic protein of the epidemic variation porcine pseudorabies virus strain, and has good immunoprotection for multiple epidemic variation porcine pseudorabies virus strains. The mouse is subjected to intramuscular injection of the anterior shin of the right lower limb, then the body can be continuously induced to generate specific neutralizing antibodies and cellular immunity at a high level.

The invention discloses an ExPEC (extraintestinal pathogenic escherichia coli) double-gene deleted strain and a vaccine prepared from the same. A construction method of the ExPEC double-gene deleted strain comprises the following steps: knocking out a relA gene and a spoT gene in an ExPEC virulent strain DE205B by a Red homologous recombination method to obtain an ExPEC relA and SpoT double-gene deleted strain DE205B[delta]relA/spoT, the strain is preserved in the China Center for Type Culture Collection, and the preservation number of the strain is CCTCC NO: M 2020610. The deleted strain has good biological safety and immunogenicity, is relatively weak in toxicity, and can be used for preparing the attenuated vaccine and preventing ExPEC infection. The attenuated vaccine prepared from the ExPEC double-gene deleted strain has an immune protective effect on ExPEC infection, and has a good toxin attacking immune protective effect.