57 results about "Orf2 gene" patented technology

The invention mainly aims to provide a porcine circovirus type 2 (PCV2) subunit vaccine and a preparation method thereof. Particularly, a baculovirus vector expression system is utilized to express a large amount of recombinant open reading frame type 2 (ORF2) protein in insect cells, so that the subunit vaccine with good immunity effect is developed. A bac-to-bac baculovirus expression system is adopted to perform whole gene amplification on the porcine circovirus type 2 ORF2 gene, and a melittin signal peptide nucleotide sequence is introduced into a terminal 5', so that the recombinant baculovirus of an open reading frame containing the melittin signal peptide nucleotide sequence and a PVC2ORF2 gene sequence is established, wherein the infected insect cell expresses the recombinant ORF2 protein with efficient and high PCV2 antigenicity; and thus the subunit vaccine containing the PCV2 recombinant ORF2 protein is prepared. The inoculation experiments of piglets show that the subunit vaccine has a good immunity protection effect.

The invention discloses a preparation method of a recombinant porcine circovirus type 2 Cap antigen, which comprises the following steps: using a yeast expression system to amplify the PCV2-ORF2 gene; then, constructing an expression engineering bacterium: inserting an amplification sequence into a yeast expression vector to form a recombinant expression vector, linearizing, and transforming into a pichia pastoris host cell so as to finish the construction of the expression engineering bacterium; and finally, carrying out secretory expression on the recombinant microzyme to obtain the recombinant porcine circovirus type 2 Cap antigen protein. The yeast expression system can carry out processing, folding and posttranslational modification on the expressed protein, so that the expressed protein has biological activity. Compared with Escherichia coli, the yeast expression system has more advantages; and compared with a mammal cell expression system, the yeast expression system is more convenient to operate. Compared with Saccharomyces cerevisiae, the pichia pastoris can not be easily subjected to hyperglycosylation, thereby facilitating the separation and purification steps. By using the yeast expression system, the invention has the advantages of simple operation process, high expression quantity and low production cost, can implement large-scale production, and is beneficial to the popularization and application of the porcine circovirus subunit vaccine.

The invention relates to a porcin circovirus type 2 (PCV2) recombinant autographa californica multicapsid nucleopolyhedrovirus construction method and a subunit vaccine preparation method thereof. The novel recombinant lucerne fork vein noctuid nuclear polyhedrosis virus construction method comprises the steps that: the main surface antigen which expresses the PCV2 is used to prepare the subunit vaccine with better immunity. Specifically, in the carrier expression system of the PCV2 recombinant autographa californica multicapsid nucleopolyhedrovirus, a hepatitis E virus open reading frame (ORF2) gene of the PCV2 is directionally inserted, so the gene efficiently expresses in insect cells and has good immunogenicity, the prepared corresponding subunit vaccine can stimulate pigs to generate the protective immunoreaction against the attack of the strong virus of the PCV2.

The invention discloses a series of N-terminal truncated PCV2-type ORF2 (Open Reading Frame 2) genes, a virus-like particle of the ORF2 gene and a preparation method of the virus-like particle, a vaccine including the truncated virus-like particle and an application of the vaccine in the prevention of post-weaning multisystemic wasting syndrome infection. The virus-like particle assembled by truncated PCV-ORF2 genes disclosed by the invention has good immunogenicity and protection. Since no denaturing agent is used in the preparation and purification process disclosed by the invention, the activity of the target protein is protected to the greatest extent.

The invention discloses a construction method and an application of a porcine encephalomyocarditis virus BD2 strain full-length infectious clone. On the basis of the constructed porcine encephalomyocarditis virus BD2 strain full-length infectious clone, an ORF2 gene of porcine circovirus is interpolated into an L gene encoding virus pilot protein, so that the encephalomyocarditis virus BD2 strain full-length infectious clone, which stably carries the ORF2 gene of the porcine circovirus 2, is successfully constructed. Both the constructed EMCVBD2 strain full-length infectious clone and the encephalomyocarditis virus BD2 strain full-length infectious clone, which stably carries the ORF2 gene of the porcine circovirus 2, can rescue infectious recombinant virus; therefore, the EMCVBD2 strain full-length infectious clone and the encephalomyocarditis virus BD2 strain full-length infectious clone, which stably carries the ORF2 gene of the porcine circovirus 2, can be applied to vaccine preparation and virus researches.

A chimeric hog cyclovirus PCV1-2 is disclosed. Its configuring method include ssuch steps as using the PVC2-ORF2 of hog cyclovirus PVC2 to replace the PVC1-ORF2 of PVC1, configuring a fragment of deficiency PCV1-ORF2 genome (pSK-PVC1 delta ORF2), and linking it to a fragment of PCV2-ORF2 genome. It can be used to prevent and treat the multi system failure syndrome of delactated piglet by PCV1-2 infectious immunizing cesarean section and deprivating foremilk.

The invention provides a restructing adenovirus of 2-type pig-ring virus and constructing method thereof, and the preventing appliances of pigs' relevant diseases caused by PCV2, including: augment and clone of PCV2 ORF2 genes; construction of recombinant shuttle vectors containing ORF2 genes; construction of recombinant adenovirus plasmid vectors; acquirement of recombinant adenovirus. Said recombinant adenovirus could determinately grow inside the bodies and continueously express ORF2 proteins to stimulate the organisms which generate neutralizing antibodies for ORF2 proteins, and offer endurance protection to pigs with promising future in the prevention and cure of PCV2, which could prevent efficiently poor quality of male-pig sperms, abortion, dead fetus, mummy and low production of female-pigs to increase the uniformity of pigs greatly.

The invention discloses a RT-RPA-lateral flow tomography kit for doubly detecting hepatitis e virus and hepatitis a virus. The kit comprises an upstream primer, an intermediate probe and a downstreamprimer which are applicable to hepatitis e virus ORF2 gene sequence in the RT-RAP amplification technology, and/ or an upstream primer, an intermediate probe and a downstream primer which are applicable to hepatitis a virus VP1 gene sequence, general agents for recombinase polymerase amplification technology, and a lateral flow tomography test strip, wherein the lateral flow tomography test stripcomprises a sample adding pad, a control line, a #1 detecting line and/ or a #2 detecting line; the control line, the detecting lines and the primers are combined with a probe label. With the adoptionof the kit, the hepatitis e virus ORF2 gene and/ or hepatitis a virus VP1 gene in a sample can be rapidly, sensitively and specifically detected on site in a non-lab environment.

The invention relates to a fourfold real-time fluorescent PCR identifying and detecting method for porcine circoviruses 1 to 4, and belongs to the technical field of biology. In accordance with ORF1 and ORF2 genes of different genotypes of PCV, specific primers and probe sequences, capable of identifying PCV1, PCV2, PCV3 and PCV4, and a probe of the PCV1-4 strains labeled with different fluorescein can be designed; each probe can be labeled with any fluorescein, and fluorescein for labeling the four probes needs to be detected by different detection passages; the primers and probes for detecting the PCV1-4 strains, or the primer and probe for identifying the PCV1-4 strains are placed in the same reaction tube, a fluorescent PCR amplification instrument is used for a real-time fluorescent PCR reaction, and the same sample is detected once, so that identification and detection on the PCV 1-4 strains can be completed.

The invention discloses a recombinant porcine pseudorabies virus (Herpesviridae) strain used for expression of porcine circovirus type II ORF2 gene. Preservation number of the recombinant porcine pseudorabies virus strain is CCTCC No.V201315. According to the preparation method, PCV2ORF2 gene is inserted into common carrier PG so as to construct transferring plasmid PGO; monolayer ST cells are inoculated with PRVTK<->/gG<->/gE<-> virus by 2h of adsorption; ST cells are transfected with plasmid PGO, wherein fusion degree of the monolayer ST cells is 80 to 90%; the recombinant porcine pseudorabies virus PGO strain is obtained by plaque purification, and is used for immunization of mouse. It is shown by results that commercial PCV2 inactivated vaccine and a group immunized by the recombinant porcine pseudorabies virus PGO strain are both capable of inducing specific humoral immune response of mouse, antibody titers of both the commercial PCV2 inactivated vaccine and the group are obviously higher than that of a group immunized by DMEM medium, and difference is significant (p<0.05). It is shown by the results of mouse lymphocyte proliferation test that specific ceullar immune response caused by the group immunized by the recombinant porcine pseudorabies virus PGO strain is more obvious than that caused by the commercial PCV2 inactivated vaccine and the group immunized by DMEM medium, and difference is obvious (p<0.05). In addition, the group immunized by the recombinant porcine pseudorabies virus PGO strain is capable of resisting severe attack by PCV2. Therefore application of the recombinant porcine pseudorabies virus strain in development of a novel PCV2 vaccine is possible.

The invention relates to a method for purifying recombinant porcine circovirus 2 type Cap protein. The recombinant porcine circovirus 2 type Cap protein is composed of ELP type elastin polypeptide genes and PCV-ORF2 genes. A preparation method of the purifying recombinant porcine circovirus 2 type Cap protein comprises the steps of expression vector construction and fusion protein expressing and purifying. Compared with an existing porcine circovirus vaccine purifying method, the method for purifying the recombinant porcine circovirus 2 type Cap protein has the advantages that preparation is easy and convenient, purifying is easy, and the price is low, and is suitable for large-scale production operation, and a foundation is provided for preparation, purification and application of subsequent recombinant porcine circovirus subunit vaccines.

The invention discloses a loop-mediated isothermal amplification (LAMP) diagnostic kit for porcine circovirus type 2. The diagnostic kit comprises 250muL of LAMPmix buffer solution, 20muL of (i)Bst(/i) deoxyribonucleic acid (DNA) polymerase, 150muL of ultrapure water, 40muL of mixed inner primer 1 and inner primer 2, of which the concentration is 40pmol/L, 40muL of mixed outer primer 1 and outer primer 2, of which the concentration is 5pmol/L, 20muL of negative control and 20muL of positive control. Two pairs of primers are designed and synthetized according to a porcine circovirus type 2(PCV-2) ORF2 gene sequence in GenBank, an LAMP diagnostic method for the porcine circovirus type 2 is built by optimization of the LAMP condition, and the sensitivity and specificity tests, and a simple and fast method is provided for rapid diagnosis of the porcine circovirus type 2 on the spot. Thus, quick quarantine needs of the basic level on the spot are met.

The invention discloses a porcine circovirus type 3 PCR detection primer pair and a porcine circovirus type 3 PCR detection kit. The primer pair includes a forward primer having a nucleotide sequence shown as SEQ ID NO:1 and a reverse primer having a nucleotide sequence shown as SEQ ID NO:2. The primer pair is used for amplifying an ORF2 gene segment of porcine circovirus type 3, and a target gene segment, which is amplified, is 408bp. The invention also provides the porcine circovirus type 3 PCR detection kit and a using method of the kit. The primer pair provided by the invention, when used for detecting the porcine circovirus type 3, has the characteristics of being high in accuracy, strong in specificity, high in sensitivity, simple, rapid and the like; and the primer pair is applicable to etiological investigation of the porcine circovirus type 3.

This invention relates to modified ORF2 gene of porcine circovirus type 2 and its application. The modified ORF2 gene is obtained by: replacing the gene coding 41 amino acids at N-termal of natural ORF2 with the gene coding signal peptide of human tissue plasminogen activator, deleting terminator of ORF2 gene, and fusing the gene coding C-terminal of ORF2 with the gene coding the transmembrane peptide of avian influenza virus HA protein. The nucleotide sequence of ORF2 gene is shown in SEQ ID No.1. This invention also relates to eukaryotic plasmid pcDNA-spORF2 Delta41TM, and host E. coli strain DH5alpha/pcDNA-spORF2 Delta 41TM (CCTCC No.M207069). The ORF2 gene and the plasmid can be used for manufacturing the vaccine for preventing post-weaning multi-systematic syndrome.

The invention relates to the field of biotechnology, in particular to a recombinant adenovirus for expressing porcine circovirus type-3 ORF2 gene and a preparation method and application of the recombinant adenovirus. The invention provides a recombinant adenovirus transfer plasmid containing a Cap protein coding gene of PCV3 and a recombinant adenovirus. According to the invention, through artificial codon optimization and signal peptide selection, high-level secretion expression of the Cap protein of PCV3 is realized, the structure and function of the obtained Cap protein of PCV3 are completely consistent with the natural state, the immunogenicity of the Cap protein is reserved to the greatest extent, and the Cap protein can be recognized by antigen presenting cells such as DC better and faster. The adenovirus vector vaccine provided by the invention has high immunogenicity and safety, can effectively activate the immune response of an organism, stimulates animals to generate high-level specific antibodies and neutralizing antibodies, and plays an excellent immune protection effect on porcine circovirus.

The invention relates to a method for efficiently expressing an ORF2 (Open Reading Frame 2) gene of a goose astrovirus soluble capsid precursor, and application of the method, and belongs to the fieldof veterinary biological products. The ORF2 gene of the goose astrovirus is divided into two-segment expression according to the difference of gene sequence functions, and expression products are independently capsid precursor (short for cap) and capsid spike (short for spike). Two segments of proteins segmented according to functional zones can be expressed by escherichia coli, baculovirus and aCHO (Chinese Hamster Ovary) expression system. Through a purification method, the capsid precursor expression protein and the capsid spike expression protein of the goose astrovirus can be obtained.The expressed capsid precursor and capsid spike can be used for preparing a subunit vaccine and an egg yolk antibody of the goose astrovirus. The subunit vaccine and the egg yolk antibody prepared bythe method disclosed by the invention have a good immune effect and treatment effect, can be used for preventing diseases caused by the goose astrovirus and have the advantages of being in low in production cost, simple in operation and good in biological safety.

The invention relates to a recombinant virus of a PCV2 codon optimized ORF1 and ORF2 tandem gene, and belongs to the biopharmaceutical field. The artificially-synthesized codon optimized PCV2 ORF1 and ORF2 gene is named YHsfORF1-2. The YHsfORF1-2 is cloned to a baculovirus expression vector pFastBacHTA to obtain a recombinant plasmid pFBHYHsfORF1-2; and the recombinant plasmid is transfected to an Sf9 cell to obtain a recombinant baculovirus vFBHYHsfORF1-2. In the recombinant baculovirus vFBHYHsfORF1-2, ORF1 and ORF2 proteins can be successfully efficiently expressed, and can form virus-like particles. The invention also relates to an application of the recombinant virus in vaccines. Animal tests prove that inactivated vaccines prepared through using the recombinant virus can stimulate the body to generate efficient immune response and have a good immune protection effect on PCV2.

The invention discloses a porcine circovirus type III SYBR Green I real-time fluorescent quantitative PCR detection primer pair and a kit. The primer pair comprises a positive primer of nucleotide sequence as shown in SEQ ID NO:1 and a negative primer of nucleotide sequence as shown in SEQ ID NO:2. The primer pair is used for amplifying a partial segment of an ORF2 gene of the porcine circovirus type III, and the size of the amplified target gene segment is 131 bp. The invention also provides a porcine circovirus type III SYBR Green I real-time fluorescent quantitative PCR detection kit and a use method of the kit. When the primer pair is used for detecting the porcine circovirus type III, the primer pair has the characteristics of high specificity, high sensitivity, high repeatability, rapidness and the like, and can be applied to etiology detection of the porcine circovirus type III.

The invention discloses a PCV2 (porcine circovirus 2) recombinant baculovirus-like particle subunit vaccine and a preparation method thereof. According to the vaccine, an insect taken as a host cell is transfected with a eukaryotic expression vector containing a PCV2 recombinant baculovirus ORF2 gene fragment. The nucleotide sequence of the PCV2 recombinant baculovirus ORF2 gene fragment is as shown in SEQ ID NO. 1, and the eukaryotic expression vector is pAcGP67-A-PCV2. According to the method, the recombinant baculovirus containing the highly immunogenic PCV2 ORF2 nucleotide sequence is constructed by a molecular biology means, and PCV2 capsid protein can be efficiently expressed by the recombinant virus in the Sf9 cell line and assembled into PCV2 virus-like particles with PCV2 surfaceantigens in a cytoplasm. A harvested virus stock solution is processed by a filtration system and inactivated, and supplemented with an adjuvant, and the PCV2 recombinant baculovirus-like particle subunit vaccine with high immunity and good safety is prepared.

The invention discloses application of recombinant adenovirus containing porcine circovirus type 2 ORF2 genes as a standard sample in nucleic acid amplification testing. The adenovirus recombination technology is used to prepare the recombinant adenovirus containing the porcine circovirus type 2 ORF2 genes, the recombinant adenovirus is used as the standard sample of the nucleic acid amplification testing (NAT), the standard sample is stable, simple to prepare and free of biosafety risks, virus particular structure of the recombinant adenovirus is generally similar to that of pig prokaryotic virus, whole-process quality control of virus DNA extraction and amplification detection can be achieved, features such as certain quantity value are achieved, and the recombinant adenovirus is suitable for the quality control of a porcine circovirus type 2 nucleic acid amplification detection method and evaluation of detection reagents and related abilities of laboratories.