356 results about "Circovirus" patented technology

The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same position. The chimeric virus advantageously retains the nonpathogenic phenotype of PCV1 but elicits specific immune responses against the pathogenic PCV2. The invention further embraces the immunogenic polypeptide expression products. In addition, the invention encompasses two mutations in the PCV2 immunogenic capsid gene and protein, and the introduction of the ORF2 mutations in the chimeric clones.

The present invention provides for a novel oil-in-water (O / W) emulsion, with increased stability in the presence of bacterial or viral suspensions, especially those concentrated and non-purified or weakly purified. The emulsion of the present invention can act as vehicle for the delivery of a pharmaceutical composition comprising at least one immunogen and, in particular, an immunogen selected from the group comprising an inactivated pathogen, an attenuated pathogen, a subunit, a recombinant expression vector, and a plasmid or combinations thereof. In one embodiment, the present invention provides for an injectable oil-in-water (O / W) emulsion comprising: (1) an aqueous solution containing an immunogen, said immunogen selected from the group comprising an inactivated Mycoplasma hyopneumoniae bacterium, an inactivated porcine circovirus type 2 (PCV-2) virus or combinations thereof; (2) a mineral oil; (3) a non-ionic lipophilic surfactant; and (4) a non-ionic hydrophilic surfactant having a low HLB value which comprises ethoxylated fatty acid diesters of sorbitan (generally having HLB value between 11 and 13). In another preferred embodiment, the present invention provides for an injectable oil-in-water (O / W) emulsion comprising: (1) an aqueous solution containing an immunogen; (2) a non-ionic hydrophilic surfactant having a high hydrophilic-lipophilic balance (HLB) value greater than 13 and less than 40, in particular HLB≧13.5, and preferably HLB≧14; (3) a mineral oil; (4) a non-ionic lipophilic surfactant; and (5) a non-ionic hydrophilic surfactant having a low HLB value (HLB value of about 9 to about 13).

Porcine circovirus-2 (PCV-2) is a recently identified agent wherein the potential spectrum of PCV-2-associated disease has been expanded by evidence of vertical and sexual transmission and associated reproductive failure in swine populations. PCV-2 was isolated from a litter of aborted piglets from a farm experiencing late term abortions and stillbirths. Severe, diffuse myocarditis was present in one piglet associated with extensive immunohistochemical staining for PCV-2 antigen. Variable amounts of PCV-2 antigen were also present in liver, lung and kidney of multiple fetuses. Inoculation of female pigs with a composition including an immunogen from PCV-2 or an epitope of interest from such an immunogen or with a vector expressing such an immunogen or epitope of interest prior to breeding, such as within the first five weeks of life, or prior to the perinatal period, or repeatedly over a lifetime, or during pregnancy, such as between the 6and 8and / or the 10and 13weeks of gestation, can lower viral titer, prevent myocarditis, abortion and intrauterine infection associated with porcine circovirus-2. In addition, innoculation of male and / or female pigs with the aforementioned compositions can be carried out to prevent transmission of PCV-2 from male to female (or vice versa) during mating. Thus, the invention involves methods and compositions for preventing myocarditis, abortion and intrauterine infection associated with porcine circovirus-2.

The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same position. The chimeric virus advantageously retains the nonpathogenic phenotype of PCV1 but elicits specific immune responses against the pathogenic PCV2. The invention further embraces the immunogenic polypeptide expression products.

The present invention relates to methods for culturing circovirus and in particular, porcine circovirus. The present invention provides compositions and methods for culturing porcine circovirus in mammalian cells expressing mammalian adenovirus E1 function.

The invention discloses porcine circovirus II-type recombinant baculovirus as well as a preparation method and application thereof. ORF2 gene is artificially synthesized by referring to a PCV2b isolated strain ORF2 gene sequence; the synthesized ORF2 gene is connected to pFBDPHmHNM1P10eGFP plasmid by adopting the plasmid as a framework vector, so that a baculovirus transfer vector pFBDPHm 30RF2 is obtained. The baculovirus transfer vector pFBDPHm30RF2 is mixed with DH10Bac escherichia coli competent cells, and the positive bacterial colony is selected to obtain a recombinant rod granule rBac-PVR30RF2; the rod granule is transferred with a sf9 cell to obtain the recombinant baculovirus QP-Ac-30RF2. The recombinant baculovirus can be used for efficiently expressing the PCV20RF2 protein and forming virus-like particles. The VLP which is expressed and packaged by the recombinant baculovirus disclosed by the invention is used for preparing inactivated vaccine, and the organism is induced to generate specific immunity response after a 28-day-aged piglet is immunized, and the pig body can be completely protected from virulent attacks of the porcine circovirus.

The invention provides a mycoplasma hyopneumoniae strain HN0613 which is isolated and identified to have better immunogenicity, and further provides a mycoplasma hyopneumoniae antigen prepared from the mycoplasma hyopneumoniae strain HN0613 and mycoplasma pneumonia pneumovax containing the mycoplasma hyopneumoniae antigen. The invention further provides a bivalent combined vaccine of porcine circovirus II and mycoplasma hyopneumoniae. The duplex combination vaccine comprises PCV (Porcine Circovirus) antigen II (inactivated porcine circovirus antigen II or PCV20 RF2 protein), inactivated mycoplasma hyopneumoniae and vaccine adjuvant. The combination vaccine can achieve the purpose of preventing the porcine circovirus disease and the mycoplasma pneumonia swine by injection once, and has a protective effect of preventing mycoplasma hyopneumoniae infection.

The invention discloses a multiplex real-time fluorescent quantitation PCR method capable of detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and hog cholera virus at the same time. The method of the invention detects that the four virus are all in good linear relations at the same time, all ten times serial dilution points of constructed normal plasmid are all in one straight line, CT value and copy number are in good linear relation, and regression analysis shows that the related coefficient of the CT value and the copy number is R<2> more than 0.99.The multiplex real-time fluorescent quantitation PCR method has the advantages of excellent specificity, sensibility and stability, which can rapidly, sensitively and differentially detect the four virus with serious harm to the economy and can be used for early diagnosis of virus infection.

The invention discloses an antibacterial antivirus Chinese medicinal composition for the animal, comprising the following main medicament ingredients: scutellaria extract, honeysuckle extract, isatis root extract and glycyrrhiza extract. the pharmaceutical composition of the invention can be made into premix, injection, oral liquid or granula, also can be made into feedstuff premix adding into the feedstuff. the chinese medicinal composition of the invention is a pure Chinese medicinal compound preparation which has the antibacterial, anti-inflammation, antivirus and immunity-reinforced functionsand wide application range, can effectively prevent and treat various livestock and poultry diseases caused by the virus and the bacteria, such as piggyyellow-white dysentery, swine plague, infectious pleuropneumonia, asthma, canker, circovirus, swine influenza, white diarrhea, newcastle disease, bursa of Fabricius, egg drop syndrome, duck serositis and the like, and has fast effect and remarkable curative effect.

The genome sequences and the nucleotide sequences coding for the PWD circovirus polypeptides, such as the circovirus structural and non-structural polypeptides, vectors including the sequences, and cells and animals transformed by the vectors are provided. Methods for detecting the nucleic acids or polypeptides, and kits for diagnosing infection by a PWD circovirus, also are provided. Method for selecting compounds capable of modulating the viral infection is further provided. Pharmaceutical, including vaccines, compositions for preventing and / or treating viral infections caused by PWD circovirus and the use of vectors for preventing and / or treating diseases also are provided.

The present invention discloses a recombinant baculovirus strain rBac / PCV2Cap (microorganism preservation number: CGMCC NO.2083) efficiently expressing Porcine circovirus type 2 Cap protein and applications thereof. The recombinant baculovirus strain rBac / PCV2Cap constructed by the present invention can efficiently express recombinant PCV2-Cap protein in insect cells, and the expressed recombinant Cap protein, which has good immunocompetence and antigenicity, can serve as a subunit vaccine used to prevent the related plague caused by Porcine circovirus type 2 infection as well as a detecting and diagnostic antigen for Porcine circovirus type 2 serum antibody.

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 belongs to the animal virus genetic engineering technique field, especially to a recombinant porcine pseudorabies virus-porcine propagate and breath complex virus-porcine circovirus gene engineering strain construction, a vaccine preparation and applications. The recombinant porcine pseudorabies virus-porcine propagate and breath complex virus-porcine circovirus gene engineering strain E001 lacks the pseudorabies virus main virulence gene TK, glycoprotein genes gG, gE and gI, does not express the functional glycoprotein gG and gE / gI of the pseudorabies virus; and simultaneously expresses a GP5m / M protein antigen of the porcine propagate and breath complex virus classical strain and the porcine propagate and breath complex virus internal variant GP5 protein and porcine circovirus ORF2. The strain can stimulate the porcine to produce protective immunity reaction for resisting pseudorabies virus-porcine propagate and breath complex virus-porcine circovirus, effectively prevent the infection of the pseudorabies virus, the porcine propagate and breath complex virus and porcine circovirus. The invention also discloses a preparation and application of a tervalence genetic engineering vaccine.

The invention provides a vaccine composition for preventing and treating porcine circovirus type 2, haemophilus parasuis and mycoplasma hyopneumoniae infection. The vaccine composition comprises an inactivated porcine circovirus type 2 antigen, inactivated haemophilus parasuis, inactivated mycoplasma hyopneumoniae and a vaccine adjuvant. The vaccine composition disclosed by the invention can realize the aim of preventing three diseases including a porcine circovirus disease, mycoplasma pneumonia, a haemophilus parasuis disease by one injection of the vaccine; the content of antigen is 1 / 2 of the content of a common single-vaccine antigen when the vaccine composition disclosed by the invention is prepared by mixing the three antigens; and compared with the existing condition that three injections of single vaccine are injected to prevent three infectious diseases, the technical scheme disclosed by the invention is economical and practical, reduces the production cost, simplifies an immune procedure and reduces the epidemic prevention cost.

The invention provides a method for efficiently preparing porcine circovirus 2 type empty capsid, which comprises the following steps: cloning different viral strain lines of antigen genes cap required by forming circovirus empty capsid and artificially reconstructed cap mutant into a domestic silkworm baculovirus carrying vector to obtain a homologous recombinant vector, recombining or transpositioning the homologous recombinant vector and parent virus DNA (deoxyribonucleic acid) in an insect cell or bacterium to obtain recombinant baculovirus, and infecting the insect with the recombinant baculovirus containing the antigen gene; and culturing the infected insect host to express the corresponding circovirus 2 type empty capsid, determining the information required by efficiently assembling the virus empty capsid by comparison and experimentation, and obtaining a recombinant viral strain line capable of efficiently expressing and assembling virus empty capsid, thereby carrying out mass production on the circovirus 2 type empty capsid. The porcine circovirus 2 type empty capsid produced by the method can be used for preparing vaccines for preventing and treating porcine circovirus disease after being subjected to primary purification.

The invention discloses an indirect ELISA diagnosing reagent box for pig II type circular virus (PCV2) antibody. There arranged with antibody detecting board in the reagent box, enzyme combination liquid, positive comparison, negative comparison, sample diluter, 10X condensed detergent, developing liquid A and B, and stopping liquid, the detecting board of the reagent box is removable 96-apertures enzyme labeling board enclosed with PCV2 reconstructed enucleation localizing signal capsid protein (dCap), the enzyme combination liquid is goat-anti-pig IgG multi-clone antibody labeled with HRP, the positive comparison is pig PCV 2 standard positive serum, the negative comparison is pig standard negative serum. The sensitivity of the invention is high, simple, and easy to be wide applied.

The invention provides a set of nucleic acids of a liquid-phase gene chip for synchronously detecting five porcine viruses, which comprise forward and reverse primers and hybrid probes for porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV) and porcine parvovirus (PPV). The invention also provides a multiplex liquid-phase chip high-flux molecular biology detection method of the five porcine viruses. According to the method, porcine virus nucleic acids in the sample to be detected are extracted to perform multiplex unsymmetric nucleic acid amplification / multiplex liquid-phase gene chip (suspension chip) combined detection, thereby synchronously and accurately detecting and identifying the five porcine viruses in the sample to be detected. The method has the advantages of high specificity, high sensitivity, high stability, high flux and high detection speed, and is simple to operate.

The invention relates to a pig breeding and breathing complex virus (PPRSV) and pig 2-type loop virus (PCV 2) series gene recombination adenovirus and vaccine in the field of high and new biotechnology. It uses PCR technology to clone the Cap protein gene into the plasmid carrier pShuttle-CMV-GP5 by open type reading rule and transforms tobacillus coli BJ5183strain with cage carrier pAdEasy-1 to capture the recombination plasmid; it uses recombination plasmid HEK293-A cell to capture recombination adenovirus and purifies it to express the recombination adenovirus rAd-Cap-GP5 of PRRSV GP5protein and PCV-2 Cap protein.

The invention discloses a bactrian camel heavy-chain (HC) variable-domain antibody resisting a porcine circovirus 2 as well as a preparation method and an application thereof, and belongs to the field of a biotechnology. A PCV2 (Porcine Circovirus Virus) inactivated vaccine immunized bactrian camel is firstly utilized and a phage display technology is utilized to establish a PCV2 immune VHH antibody base; and a high-specificity variable domain antibody of heavy chains of HCAbs (VHH) which has a good affinity with the PCV, particularly a PCV2 virus antigen; and the antibody disclosed by the invention has an amino acid sequence shown as SEQ (sequence) ID NO.1 or an amino acid sequence which is shown as SEQ ID NO.2 and has the sequence isogeny being more than 95%. The PCV2 heavy-chain variable-domain antibody disclosed by the invention has the very important meanings on researching porcine circovirus, particularly researching a porcine circovirus 2-type infection mechanism and establishing rapid antigen detection with high sensitivity and specificity or diagnosing a reagent.

The invention discloses a preparation method for a PCV-II Cap protein monoclonal antibody, an antibody and application. The invention adopts ultracentrifuged and purified PCV-II as an immunogen to immunize a BALB / c mouse by the conventional method, takes spleen cells of the immunized BALB / c mouse to fuse with SP2 / 0 cells, obtains two strains of hybridoma cells secreting the PCV2-Cap protein monoclonal antibodies by indirect ELISA screening, respectively names the two strains of hybridoma cells as 8-60 and 10-48, identifies biological characteristics of the two strains 8-60 and 10-48, and usesthe two strains 8-60 and 10-48 as the first antibodies to establish an indirect immunofluorescence diagnostic method. The result of the indirect immunofluorescence diagnostic method is basically consistent with that of the PCR diagnostic method, and the positive and negative coincidence rates are respectively 93.75 percent and 100 percent so as to provide reference for preventing and treating theporcine circovirus disease.

The present invention relates to methods and compositions for vaccinating pigs against porcine circovirus associated diseases.

A Chinese patent medicine for treating such diseases of domestic animals as gyrate virus disease, streptococcicosis, bilharziasis, feverous enteritis, dysentery, etc. Its application method is also disclosed.

The invention discloses an RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting a high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof. The kit comprises a pair of primers and a probe, the sequences of the primers are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the probe is shown as SEQ ID NO.3. It is proved through experiments that the kit can detect adverse effects of the high-pathogenicity porcine reproductive and respiratory syndrome virus (HP-PRRSV), a hog cholera virus, a C-type porcine reproductive and respiratory syndrome virus, a porcine circovirus type II, a porcine pseudorabies virus and a foot and mouth disease virus in a specificity mode. It is proved through experiments that the kit can detect out templates of at least 70 copies at the temperature of 40 DEG C on the condition of 20 min amplification, and the conformity between the kit and RT-qPCR is high. This shows that the kit can detect HP-PRRSV fast, efficiently and sensitively and provides an effective technological means for differential diagnosis of HP-PRRSV.

The invention discloses a PCR (Polymerase Chain Reaction) primer for detecting and identifying porcine circovirus 3 (PCV3) and a detection method and a detection kit. A pair of PCR primers for detecting the PCV3 is designed by performing preference comparison with genomic sequences of the PCV3 according to a conserved sequence on a cap protein of the PCV3. Sequences of the forward and reverse primers PCV3-F / R of the PCR are sequentially shown as SEQ ID NO.1 to SEQ ID NO.2. The invention further provides a PCR detection method and kit for the PCV3. The primer and the detection method disclosed by the invention are capable of rapidly, conveniently and efficiently detecting and identifying the PCV3, the specificity is high, the sensitivity is high, and the detection method is simple and high-efficiency in operation, is convenient for clinical detection and convenient for developing epidemiological investigation and has great application prospects.

The invention relates to a vaccine of the truncated Cap protein of porcine circoviruses type 2 and a preparation method thereof. The preparation method comprises: firstly, cloning the Cap protein gene, in which 41 amino acid residues in an N terminal is removed, of the porcine circoviruses type 2 into a plasmid pKK-233-2 to obtain a recombinant expression plasmid pKK-233-2-Cap; and transforming Escherichia coli DH5 alpha by the recombinant expression plasmid to obtain recombinant genetic engineering bacteria DH5 alpha / pKK-233-2-Cap. The addition of IPTG or lactose for induction is avoided, the recombinant genetic engineering bacteria express the truncated Cap protein constitutively, the truncated Cap protein is separated and purified, and an adjuvant is added to obtain the vaccine of the truncated Cap protein of porcine circoviruses type 2. The Cap protein can also be used for preparing reagent for diagnosing related diseases caused by the porcine circoviruses type 2.

The invention relates to a method for preparing a porcine circovirus type 2 colloidal gold antibody fast test strip. In the method, a porcine circovirus type 2 ORF2 protein is expressed by utilizing genetic engineering, and the test strip is prepared by the principle of enzyme-linked immunoassay and membrane chromatography to fast test antibodies in the blood or serum of pigs. The test strip can be widely used for clinically testing porcine circovirus diseases, has no cross reaction with other viruses, and obtains test results 98.63 percent and 95.83 percent consistent with those obtained by the two methods of the ELISA and neutralization test. Compared with the ELISA, a recombinant antigen immune colloidal gold has the remarkable advantages of high security, no need of culturing the viruses per se, the avoidance of virus spread caused by the operation of the viruses, large-batch preparation, simple process, low production cost, stable and homogeneous antigen components, simple, convenient and labor-saving operation, no apparatus, high detection result specificity, high repeatability, the short time of 15 minutes for the whole test, simple, convenient, fast and accurate operation, high sensitivity, intuition and easy result judgment.

The invention provides a kluyveromyces marxianus engineering bacterium obtained from recombinant expression of porcine circovirus type II capsid protein; and porcine circovirus type II virus-like particles are obtained by cloning porcine circovirus type II capsid protein genes into an expression vector and conducting recombinant expression in kluyveromyces marxianus. The invention also provides a preparation method of a porcine circovirus type II virus-like particle vaccine, wherein the preparation method comprises the following steps: preparing the porcine circovirus type II virus-like particles through high-density fermentation of the recombinant kluyveromyces marxianus, and then conducting separation and purification so as to prepare the vaccine. The porcine circovirus type II virus-like particle vaccine provided by the invention, which can generate a high-level serum IgG antibody just by implementing injection immunization once, has the advantages of being good in immunizing effect, high in safety, simple in culture operation, high in yield, low in production cost and the like, and the porcine circovirus type II virus-like particle vaccine can achieve large-scale enlarged production; and the porcine circovirus type II virus-like particle vaccine can be used for relieving and preventing related clinical symptoms caused by the infection of porcine circovirus type II (PCV2).

The invention provides a gene chip detection method for a classical swine fever virus (CSFV), a porcine circovirus virus type 2 (PCV-2), a porcine reproductive and respiratory syndrome virus Europe type (PRRSVE) and a porcine reproductive and respiratory syndrome virus America type (PRRSVA). A gene chip is shown as a probe sequence in the table of the specification. The invention discloses a method for simultaneously detecting three diseases, namely classical swine fever (CSF), a porcine circovirus type 2 (PCV-2) and a porcine reproductive and respiratory syndrome (PRRS) by using the gene chip. By the method, the problems that the conventional detection technology is time-consuming and labor-consuming and has low specificity, or only can be used for detecting a single disease are solved. All probes provided by the invention are synthesized into a connecting amino group at a 5' end, a poly T connecting arm with the length of 15 bp is provided, and a result shows that fixing efficiency is relatively high. By a polymerase chain reaction (PCR) amplification technology with mark primers, various kinds of fluorescent mark deoxyribonucleic acid (DNA) complementary to corresponding probescan be amplified at a time, and the high specificity and sensitivity of nucleic acid hybridization in a solid-liquid phase can be ensured. In addition, by a mark method, detection time is greatly shortened, the cost of chip detection is lowered, and the detection method is more suitable to be popularized in clinical application.