461 results about "Recombinant baculovirus" patented technology

Provided are compositions and methods of use for insect cells comprising baculovirus encoding non-surface expressed proteins and peptides. The claimed invention particularly relates to compositions comprising insect cells containing baculovirus that express cytokines. Such compositions may be administered by, for example, direct intratumoral injection into tumors in mammals, resulting in tumor reduction or recission. Another aspect of the claimed invention concerns methods of promoting resistance to the reoccurence of tumors in mammals who have undergone such tumor recission. In a specific aspect of the claimed invention, the mammals are human subjects presenting with various forms of cancer.

Disclosed and claimed is a new insect cell line, Sf900+, ATCC CRL-12579. The insect cell line was established from Lepidoptera, Noctuidae, Spodoptera frugiperda Sf-9 (ATCC CRL-1711) through multiple rounds of limiting dilution and selection in a serum-free insect medium supplemented with added human insulin. The insect cell line is useful in BEVS or as an adjuvant and has many characteristics and advantages. Also disclosed and claimed are recombinant proteins from recombinant baculovirus expression in insect cells such as Sf900+ cells, for instance, HA, NA, EPO, CD4, CEA, and thrombospondin.

Disclosed and claimed is apparatus and methods for the growth of cells to high density, products therefrom and uses thereof. Also disclosed and claimed is the use of this method for the growth to high-density insect cells, such as the Spodoptera frugiperda Sf900+ cell line (ATCC: CRL 12579). Further disclosed is the infection of Sf900+ cells at high cell density with wild type and recombinant baculoviruses to produce baculovirus and DNA or gene or expression products.

Snow Mountain Virus (SMV) belongs to the Norovirus genus of the Caliciviridae family. SMV is a genogroup II (GII) reference strain of human enteric caliciviruses associated with epidemic gastroenteritis. The positive sense RNA genome sequence of SMV was determined to be 7,537 nucleotides in length excluding the 3′ polyadenylated tract. The genome is organized into three open reading frames. Pairwise sequence alignments showed SMV ORF1 is highly conserved with other GII noroviruses, and most closely related to GII strains Melksham and Hawaii viruses. Comparative sequence analyses showed the SMV is a recombinant norovirus. VP1/NP2 proteins assembled into virus-like particles (VLPs) when expressed in insect cells by a recombinant baculovirus. Characterization of one clone that expressed VP1 but failed to assemble into VLPs, identified histidine residue 91 as important for particle assembly.

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 belongs to the technical field of biological vaccine preparation, and particularly relates to a porcine epidemic diarrhea (PED) recombinant baculovirus gene engineering subunit vaccine, a preparation method and an application thereof. According to the present invention, S1 gene and M gene of the current new PEDV epidemic strain are selected as reference sequences, a baculovirus expression system is adopted to express S1 protein or partial S1 protein and M protein, and the obtained recombinant protein is prepared into a subunit vaccine for effectively controlling PED occurrence; with the PED recombinant baculovirus gene engineering subunit vaccine produced by using the method, the defect of the current PEDV traditional vaccine is solved; and the PED recombinant baculovirus gene engineering subunit vaccine can be used for prevention and treatment of PEDV infections and related diseases caused by PEDV, and can further be used for preparation of coating antigen of PEDV detection antibody ELISA kits.

The present invention discloses a method for assembling foot-and-mouth disease virus empty capsids in insect cells via the alteration of acid-resistance. The method for assembling foot-and-mouth disease virus empty capsids in insect cells includes the following steps: (1) the altered P12A gene and the non-structural protein gene 3C of foot-and-mouth disease virus are introduced into bacteria via baculovirus vectors for recombination to produce recombinant rhabdovirus A; (2) the DNA of the recombinant rhabdovirus A is used to transfect the insect cells, so that the foot-and-mouth disease virus empty capsids are obtained. The method assembles the integral foot-and-mouth disease virus empty capsids in the insect cells for the first time, lays a foundation for the research and the development of gene-engineered subunit vaccines and novel diagnostic reagents.

The invention discloses porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as a preparation method and application thereof. Sequences of VP0, VP1 and VP3 genes are artificially synthesized by referring to an FMDV (Foot And Mouth Disease Virus) O-type epidemic strain gene sequence; the VP0, VP1 and VP3 genes are connected to pFBDPHmHNM1P10eGFP plasmid by adopting the plasmid as a framework vector, so that a baculovirus transfer vector pFBDPHmVP013 is obtained. The baculovirus transfer vector pFBDPHmVP013 is mixed with DH10Bac escherichia coli competent cells, and the positive bacterial colony is selected to obtain a recombinant shuttle vector Bacmid; the shuttle vetcor Bacmid is transferred with a sf9 cell, and the recombinant baculovirus QP-Ac-FVLP is obtained by collecting the cell supernatant. The recombinant baculovirus can be used for efficiently expressing FMDVVP0, Vp1 and Vp3 proteins and forming virus-like particles. And the virus-like particles are used for preparing subunit vaccine, so that the organism is induced to generate specific immunity response after the mouse is immunized.

The invention discloses a univalent and bivalent gene engineered subunit vaccine for preventing Enterovirus 71 (EV71) and Coxsackie virus A16 (Cox.A16) of hand-foot-and-mouth disease, and a preparation method thereof. The preparation method comprises the following steps: respectively obtaining recombinant baculovirus Bac-EV71-P1-3CD and Bac-Cox.A16-P1-3CD by gene engineering means, respectively efficiently coexpressing similar SeQ ID No.1 EV71 P1 and Se Q ID No.2 Cox.A16 P1 and 3CD proteins in insect cells, and respectively self-assembling into EV71 VLP and Cox.A16 VLP; establishing and verifying a vaccine strain third-stage seed lot library; culturing cells; inoculating and propagating virus; lysing the cells, ultra-filtering and purifying virus suspension; and further preparing the univalent and bivalent vaccine. The vaccine has good application prospect for preventing the hand-foot-and-mouth disease.

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 relates to a 2 type subunit vaccine for a porcine circovirus as well as a preparation method and application thereof. A recombinant bacilliform virus contains double promoters (a polyhedrin promoter and a P10 promoter), a coding gene of a Cap protein with double copying can be expressed, and the expression efficiency of the protein is obviously enhanced; moreover, the Cap protein expressed by an inserted foreign gene does not contain an excess sequence, virus-like particles (VLPs) can be effectively formed, and the immunogenicity of an expressed protein is enhanced; furthermore, a produced antigen has high content; and according to the 2 type subunit vaccine for the porcine circovirus, which is disclosed by the invention, the productivity ratio and the quality of a viral protein of the 2 type subunit vaccine for the porcine circovirus are obviously enhanced, and a prepared vaccine composition has the advantages of stable and persistent immune effect, high safety and the like.

The invention provides a method for making rabies virus antigen. The method comprises the following steps that: the antigen gene of rabies virus or the combined expression combination of the antigen gene is respectively cloned in a baculovirus carrier so as to obtain a transfer expression carrier; the transfer expression carrier and baculovirus undergo cotransfection so as to carry out homologous recombination or transposition, thereby obtaining recombined baculovirus; the recombined baculovirus is used to infect insect host and cell; the infected insect host is cultured to express corresponding rabies antigen; and the expressed antigen is ingathered and purified so as to obtain rabies virus antigen. The method adopts a baculovirus expression system to make safe and efficient rabies virus antigen in a domestic silkworm bioreactor; moreover, due to having extremely high safety, the made antigen can be directly used to make injection vaccine and oral vaccine used for animal immunization. The method can substantially reduce the production cost of rabies virus antigen, and has the advantages of safety, high efficiency, less energy consumption and low cost, etc.

The invention discloses a method for expression of a PCV 2 (Porcine circovirus type2) Cap protein by a pFast Bac Dual baculovirus. The method comprises the following steps of: amplifying a gene fragment of an encoded PCV 2 Cap protein with a His tag; connecting the gene fragment to a pFast Bac Dual plasmid so as to obtain a recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2; transforming Escherichia coli DH10Bac with the recombinant transfer plasmid, and carrying out blue-white selection to obtain a recombinant shuttle vector Bac-p10-ORF2-pH-ORF2; transfecting an insect cell with the recombinant shuttle vector so as to obtain a recombinant baculovirus Ac.Dual-Cap; and poisoning the insect cell with the recombinant baculovirus, performing cultivation, then collecting the insect cell, and purifying an expression product so as to obtain the recombinant Cap protein. The method disclosed in the invention solves the problem of low expression level of the Cap protein in eukaryotic cells. The recombinant Cap protein in the invention is designed with a His tag, thus being beneficial to the follow-up purification. And the recombinant Cap protein has biological activity superior to that of a Cap protein expressed by a prokaryotic expression system, thus being applicable to establishment of epidemiological diagnosis methods and reseach as well as development of PCV2 subunit vaccines.

The invention discloses a SARS vaccine and preparing method, which comprises the following steps: 1) constructing external baculoviral surface display carrier of S protein of SARS coronary virus; 2) transmitting the carrier into susceptive cell with baculoviral genome plasmid Bacmid; obtaining recombinant baculoviral genome plasmid; 3) using the plasmid to infect insect cell; purifying the recombinant plasmid; obtaining the product.

A method for expressing antigens of foot-and-mouth disease virus in insect in vivo by using re-combinant baculovirus is disclosed, which comprises: cloning different genes combination of foot-and-mouth disease virus into baculovirus carrying vector to construct transfer vector, transfecting baculovirus with the transfer vector to proceed DNA recombination, and thus to get recombinant baculovirus, infecting insect host with the recombinant baculovirus, culturing the infected insect host to express the antigens of foot-and-mouth disease virus, and then collecting and purifying the expressed antigens. The preferred genes, baculovirus carrying vector, baculovirus and insect host are selected from P1-2A3C, ORF and VP1 genes of foot-and-mouth disease virus as shown in SEQ ID NO 1., 3 or 5, pVL1393, Bombyx mori Bm-NPV-ZJ8 and larvae or pupa of Bombyx mori, respectively.

The invention relates to the field of virology, in particular to a recombinant baculovirus which is manually modified and synthesized and contains a main immunogenic gene HA-NA-M1 of an influenza A H1N1 virus. The strain QP-Ac-HNM1 belongs to the baculovirus (Baculovirus) and is preserved in the China Center for Type Culture Collection (CCTCC) with the preserving number of CCTCC-V200912. The recombinant virus is capable of synchronously expressing the HA and NA of the influenza A H1N1 virus and M1 proteins to form virus particles which can be used for developing vaccines so as to prevent human beings and swine from being infected with the influenza A H1N1 virus.

A recombinant genome comprising polynucleotides encoding at least two additional molecular chaperones and a protein of interest, recombinant baculovirus vectors providing molecular chaperones and a method for producing a foreign protein using said genomes and vectors.

The invention discloses a recombinant virus for expressing swine fever virus E2 gene, and a preparation method and application thereof. According to the preference of the codon, synthetic CSFV 2.1-type immunogenic gene E2 and 1.1-type E2 gene are respectively arranged in baculovirus pH and p10 promoters to be expressed so as to obtain a recombinant baculovirus (CCTCC NO:V201338). The exogenous gene expression level is enhanced. After being used for immunizing Japanese white rabbits and pigs, the subunit vaccine prepared by the E2 protein expressed by the recombinant baculovirus can induce the organism to generate specific immune reaction and protect the organism from the attack of the swine fever virus; and differential diagnoses can be utilized to distinguish the vaccine immunized animal from the wild virus-infected animal, thereby being beneficial to eradication and purification of the eradicate swine fever virus in the pig farm.