122 results about "Avian leukosis viruses" patented technology

The invention discloses double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting an avian leukosis group specific antigen. The kit comprises an enzyme plate coated by a monoclonal antibody which is secreted by a hybridoma cell strain the preservation serial number of which is CGMCC (china general microbiological culture collection center) No.5961. The invention also discloses a double-antibody sandwich ELISA method which is established by utilizing the monoclonal antibody and is capable of rapidly and effectively detecting an ALV (avian leukosis virus). In the double-antibody sandwich ELISA method, the monoclonal antibody prepared by pronucleus expressive HLJ09mdj-1p27 albumen is utilized as a peridium antibody, and antibodies prepared by p27 are utilized as a detection antibody. According to the invention, the minimum detection amount of the p27 is 1.25 ng/ml, the method is not reacted with the common virus of birds, and the specificity is good. The method is utilized to detect egg white and an anus swab sample, and the coincidence rate is respectively 96.5% and 88.9% compared with a PCR (polymerase chain reaction) method; and the result proves that the method has the advantages of convenience, celerity, differentia, sensitivity and the like, and is useful for the detection and population purification of the ALV.

PCT No. PCT/US96/07370 Sec. 371 Date Nov. 28, 1997 Sec. 102(e) Date Nov. 28, 1997 PCT Filed May 22, 1996 PCT Pub. No. WO96/37625 PCT Pub. Date Nov. 28, 1996Recombinant avian sarcoma leukosis virus (ASLV)-derived retrovirus vectors having an expanded host range are described. The host range is expanded by the replacement of the ASLV envelope gene by an envelope gene from a virus capable of infecting both mammalian and avian cells. The resulting recombinant ASLV-derived retroviral vectors can replicate efficiently in avian cells, infect both avian and mammalian cells in high titer, and are replication-defective in mammalian cells. Thus, they are quite safe and advantageous for use in gene therapy and vaccines.

The invention relates to a kit for detecting the specific antibody of the A/ B/ J subgroup of an avian leukosis virus and a detection method thereof, and belongs to the field of molecular biology. Thekid includes a luminescent microporous plate, a sealing film, a receptor microsphere I, a receptor microsphere II, a donor microsphere, a calibrator and an analytical buffer solution; the receptor microsphere I is marked with the His-cENV protein of the avian leukosis virus A/ B subgroup, and the receptor microsphere II is marked with the His-gp85 protein of the avian leukosis virus J subgroup; the donor microsphere is marked with an rabbit-anti-chicken second-antibody. The detection method includes the steps of firstly, preprocessing a collected sera sample; then, adding the receptor microsphere I, the receptor microsphere II and the sera sample into the luminescent microporous plate as required, and detecting an optical signal value under 680 nm exciting light. The method achieves the detection on the A/ B/ J specific antibody of the avian leukosis virus, and has the advantages of no washing, simple operation, sensitive detection, high specificity and strong stability.

The invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity and to methods of producing, amplifying or sequencing nucleic acid molecules using these compositions or polypeptides, particularly at temperatures above about 55° C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and use of such nucleic acid molecules to produce desired polypeptide. The invention also relates to methods for producing Avian Sarcoma-Leukosis Virus (ASLV) RT subunits, in particular, Avian Myeloblastosis Virus (AMV) RTs, to isolated nucleic acid molecules encoding ASLV RT subunits, and to ASLV RT subunits produced by these methods. The invention further relates to nucleic acid molecules encoding recombinant RT holoenzymes, particularly ASLV RTs, methods for producing these RTs and to RTs produced by these methods. The invention also relates to kits comprising the compositions, polypeptides, and ASLV RTs of the invention.

The invention belongs to the cross technical field of molecular immunology and virology, and particularly relates to a hybridoma cell based on hybridoma type leukosis virus subgroup J isolated strain with preservation number of CGMCC (China General Microbiological Culture Collection) No. 5018, a high-specificity monoclonal antibody of avian leukosis virus subgroup J (ALV-J) surface protein (SU) secreted by the hybridoma cell and a preparation method of the monoclonal antibody. By using the high-specificity monoclonal antibody prepared by the invention, a technical support is provided for diagnosis, prevention and treatment of avian leukosis virus subgroup J disease, and the economic loss caused by avian leukosis virus subgroup J disease is effectively reduced.

The invention discloses an anti-avian leukosis virus p27 protein monoclonal antibody, a gold-colloidal strip containing the same, and application. The anti-avian leukosis virus p27 protein monoclonal antibody is generated by secretion of a hybridoma cell strain with a preservation number of CGMCC NO. 11089 or a hybridoma cell strain with a preservation number of CGMCC NO. 11090. The monoclonal antibody is high in titer to p27 protein and good in specificity, and therefore can be used for preparing a kit and the gold-colloidal strip for detecting an avian leukosis virus. A preparation method of the monoclonal antibody provided by the invention is simple, and an antibody purification process is simple, and the efficiency is high, and the cost is low. As the gold-colloidal strip prepared by the anti-avian leukosis virus p27 protein monoclonal antibody provided by the invention is adopted to detect the anti-avian leukosis virus, the specificity is strong, and the operation is simple, and convenience, speediness and simplicity are realized, besides, a special instrument and equipment are not needed, and professional training is not needed, and a result is clear and easy to recognize; the operation is simple and convenient; the popularization is easy, and the antibody is more suitable for real-time detection.

The invention provides an avian leukosis double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) antigen detection kit. The kit comprises ELISA plates which are coated on anti-avian leukosis virus (ALV) p27 monoclonal antibodies, enzyme labeling anti-ALVp27 monoclonal antibodies and the like. Antibody capturing and antibody detecting are respectively conducted aiming at different antigenic determinants of p27 protein; anti-ALVp27 monoclonal antibodies coated by the ELISA plates are obtained through secretion of hybridoma cell strain ALVP27-5D3, and the enzyme labeling anti-ALVp27 monoclonal antibodies are obtained through secretion of hybridoma cell strain ALVP27-4F12. The avian leukosis double-antibody sandwich ELISA antigen detection kit is easy, convenient and fast to operate, can be used in detecting of all subgroup virus of ALV, and is suitable for all levels of veterinarian departments in the basic level and rapid and mass screening detecting of the avian leukosis of leaving and entering the country. The avian leukosis double-antibody sandwich ELISA antigen detection kit has the advantages of being low in cost, notable in economical benefit, and wide in application prospect.

The invention discloses a typing method of resistance for resisting a subgroup A avian leukosis virus by quality chickens, which adopts an SNP locus at the 619th site of a TVA gene sequence as a research object; PCR-RLFP analysis is performed to detect whether mutation occurs at the SNP locus, and also to detect whether 4 bases are inserted into SNP loci at the 305th-308th sites of the TVA gene sequence; the two detection results are combined together to determine whether the detected chicken is a genetic resistance type or a genetic susceptibility type. The resistance typing method of the invention performs detection from the source, and omits troubles of later-stage detection; the detection method is rapid, accurate, strong in purposiveness, and simple in operation, has practice significance, and can realize rapid selection of chicken genetic characters and improvement of disease resistance of strains.

The invention relates to a J subset avian leukosis virus rapid detection test paper card which comprises a lining board; a sample pad, a golden-standard pad, a cellulose nitrate membrane and a water-absorbing pad are disposed on the lining board; the invention is characterized in that the golden-standard pad is coated with colloidal gold labelled monoclonal antibodies of mouse anti-J subset avian leukosis virus gp85 protein; the cellulose nitrate membrane is coated with a detection line formed by rabbit anti-J subset avian leukosis virus polyclonal antibodies and a quality control line formed by rabbit anti-mouse IgG polyclonal antibodies. The advantages of the invention are that: the detection method of the J subset avian leukosis virus rapid detection test paper card of the invention can rapidly detect whether a chicken body is infected by or carries J subset avian leukosis viruses, and has the advantages of rapid reaction, high sensitivity, strong specificity, simple operations, suitability for near-the-pen detection, economy and practicality.

The invention discloses a loop-mediated isothermal amplification (LAMP) reaction primer for detecting subgroup-A avian leukosis virus (ALV-A). The primer consists of a pair of outer primers, a pair of inner primers and a pair of ring primers, wherein the outer primers are shown in SEQ ID NO.1 and SEQ ID NO.2; the inner primers are shown in SEQ ID NO.3 and SEQ ID NO.4; and the ring primers are shown in SEQ ID NO.5 and SEQ ID NO.6. On the basis of the designed LAMP primer, an LAMP detection method for ALV-A subtypes is successfully established. By means of the method, the ALV-A can be distinguished from ALV-J, ALV-B, ALV-C and ALV-E, and is not subject to nonspecific reaction together with other common poultry infectious diseases. The ALV-A LAMP detection method established in the invention ensures high accuracy, high sensitivity and strong specificity.

The invention relates to a method for diagnosing avian leukosis virus subgroup J of sicken chicken flocks and belongs to the technical field of molecular biology. According to the method, C type retrovirus characteristics of the avian leukosis virus subgroup J are taken as a breakthrough point, on the theoretical basis of identifying a JL-2 strain of the avian leukosis virus subgroup J, specific primers are utilized to clone gp85 fragments of the virus strain, an expression vector PGEX-6P-1/gp85 category is constructed, an RFLP method is used to detect the genotype of NRAMP1 genes, the enzyme digestion reaction of the fragments where PCR amplification sites are located is observed, according to the research, it not only is indicated that that cocks carrying T allele have higher resistance to avian leukosis virus subgroup J, but also is found that the specificity diagnosis method established by gp85 is a powerful technical means for detecting specificity of ALV-J and timely purifying populations.

The invention provides a viral self-inactivating (SIN) vector on the basis of the avian sarcoma leukosis virus (ASLV) as well as a split-packaging system comprising in addition to the SIN vector a first helper plasmid serving for the expression of the viral fusion protein gag-pol and a second helper plasmid serving for the expression of the retroviral envelope protein (env). The first and second helper plasmid, for example contained in a packaging cell line or transiently transfected, serve for the generation of non-replicating (RCR-incompetent) viral particles containing RNA having a SIN LTR according to the invention at the 3′ terminus, wherein the RNA can have a therapeutically effective section which e.g. is denoted a transgene. This 3′ SIN LTR contains an extensive deletion of the U3 region which in the course of the reverse transcription is copied into the 5′ LTR. In addition, in the SIN vector all coding regions of ASLV as well as the retroviral splice donor site are removed.

The invention relates to a construction method and application of a DNA (Deoxyribonucleic Acid) vaccine for avian leukosis virus subgroup J. A recombinant eukaryotic expression plasmid pcDNA3.1-Fc-env of an Fc fragment gene for expressing chicken immunoglobulin G and an envelope protein (env protein) gene of the avian leukosis virus subgroup J is constructed; transient transfection and indirect immunofluorescence assay prove that the pcDNA3.1-Fc-env can be accurately expressed in a 293T cell; a great number of plasmids are extracted, purified and quantified to 1mg/ml, then, the recombinant plasmids are used for immunizing mice, each mouse is immunized three times, 100mu g of recombinant plasmids are used in each immunization, and one immunization is carried out every two weeks; the level of an ALV-J env protein-specific antibody in serum is detected to show that the pcDNA3.1-Fc-env has the effect of preventing the avian leukosis virus subgroup J.

The invention discloses a triple Polymerase Chain Reaction(PCR)kit for simultaneously identifying endogenous and exogenous avian leukosis viruses, comprising DNA polymerase, d NTP, primers, a PCR buffer and double distilled water. The invention is characterized in that the primers consist of three pairs of primers, wherein the sequences of a first pair of the primers are shown as SEQ ID NO: 1 and SEQ ID NO: 2, the sequences of a second pair of the primers are shown as SEQ ID NO: 3 and SEQ ID NO: 4, and the sequences of a third pair of the primers are shown as SEQ ID NO: 5 and SEQ ID NO: 6. The invention has the advantages that by using the multiple PCR kit of the invention, whether the infection of the avian leukosis viruses occurs is simultaneously detected at the same reaction system and the endogenous and exogenous ALV can be identified, thereby saving time and reagents and providing an effective tool for rapid and early diagnosis on chicken flocks or chicken embryos; and the triple PCR kit has high specificity and sensitivity.

The present invention provides primers and a detection kit for avian leukosis J subgroup virus PCR detection. According to the present invention, PCR detection primers are designed by analyzing avian leukosis J subgroup virus env gene, wherein nucleotide sequences of the primers are represented by sequence lists SEQ ID No.1 and SEQ ID No.2; and the primers have good specificity, and the detection method has characteristics of rapidness, simpleness, high accuracy and good sensitivity so as to provide an excellent detection method for avian leukosis J subgroup virus identification. The present invention further provides a detection kit for the avian leukosis virus, wherein the detection kit comprises a primer pair represented by the sequence lists SEQ ID No.1-2.

The invention provides a replication enhancer of avian leukosis virus subgroup J; the replication enhancer of avian leukosis virus subgroup J is collagen triple helix repeat containing 1 which can be used as the virus replication enhancer to directly, significantly and continuously promote ALV-J replication. When the virus is harvested, the viral load amount increases by two or more times as compared with a control group, and the replication enhancer has no toxic or side effects to cells. A CTHRC1 recombinant plasmid is further constructed to promote ALV-J replication, so that the CTHRC1 can be continuously expressed in the cells, ALV-J replication is continuously promoted, and the expression quantity is improved.

The invention discloses a avian leukemia double plate agglutination antigen of chicken pullorum disease-J subgroup and a preparation method thereof. The method includes constructing a plasmid vector displaying avian leukemia specific antigen GP85 of subgroup J on the surface, and transferring it into the strain for preparing chicken pullorum antigen to construct Salmonella pullorum which can display GP85 on the surface. The Salmonella pullorum can simultaneously express the surface antigen of Salmonella pullorum and the specific antigen of avian leukosis virus subgroup J through IPTG, and canbe used for preparing the double plate agglutination antigen. The double plate agglutination antigen can be used to detect two kinds of epidemic antibodies at the same time by one reaction. The operation is simple, the specificity is strong, the detection time and the detection cost are obviously saved, and the double antigen can be used for rapid detection of fence edge.

The invention discloses an RT-LAMP primer set and kit for a subgroup K avian leukemia virus. The RT-LAMP detection primer set for rapidly detecting the subgroup K avian leukemia virus comprises a pair of external primers and a pair of internal primers, and is designed according to a gp85 region of a subgroup K avian leukemia virus sequence. The RT-LAMP detection primer group for the subgroup K avian leukemia virus obtained by screening can specifically amplify ALV-K, and the intraspecies and interspecies specificity is very good; at least 8.4 * 10<4> copies/mu L of nucleic acid can be detected in a reaction, and the RT-LAMP detection method of ALV-K is simple in operation, low in cost, and short in time-consumption, and is suitable for rapid clinical detection of ALV-K.

The invention discloses a CQD (Carbon Quantum Dot) adjuvant and a vaccine containing the same, i.e., each single part of vaccine agent contains 200 to 400mug of the CQD adjuvant. The phenomenon that CQDs can be used as an excellent adjuvant for resisting ALV-J (avian leukosis virus subgroup J) is found for the first time; researches find that the CQDs prepared by the invention have no cytotoxicity, cannot induce reduction of the cell activity, are good in biocompatibility, and are safe and reliable to use in the immunizing dose range. The vaccine prepared by using the CQDs as the adjuvant, which is disclosed by the invention, with respect to a vaccine prepared by a Freund's adjuvant, can more effectively induce the generation of a high antibody titer to carry out immune protection and provide a more lasting protective immune response.

The invention discloses a test strip used in one-step rapid detection of Marek's disease virus (MDV) and subgroup-J avian leukosis virus (ALV-J). The test strip comprises a non-absorbing supporting layer, and an absorption layer adhered to the supporting layer. The absorption layer is formed by sequentially spliced components of an absorption fiber layer, a gold-labeled antibody fiber layer, a cellulose film layer, and a water absorption layer. The cellulose film layer is marked with anti-goat IgG or anti-mouse IgG control blot, and a detection blot comprising anti-MDV and anti-ALV-J antibody. The anti-MDV and anti-ALV-J antibody is an anti-MDV and anti-ALV-J antibody monoclonal or polyclonal antibody. An anti-MDV and anti-ALV-J mixed polyclonal antibody or monoclonal antibody marked by colloidal gold and corresponding to the detection blot is adhered to the gold-labeled antibody fiber layer. The test strip provided by the invention has the advantages of high detection specificity, high sensitivity, simple operation, fast detection, and intuitive result. The test strip is suitable for MDV and ALV-J virus on-site rapid combined detection, and can be used in identification and diagnosis of single infection or mixed infection of the two viruses. The test strip can be widely applied, and is suitable for popularization.

The invention relates to a method for efficiently amplifying subgroup J avian leukosis virus (ALV). According to the method, chicken hepatocytes can be used for efficiently amplifying the subgroup J ALV, the characteristic of high titer virus can be produced earlier by using the chicken hepatocytes, the separation and detection time of the subgroup J ALV can be shortened, the pathogen can be detected earlier, and the sensitivity of a subgroup J ALV detection method is improved. When subgroup J ALV infection MOI is 0.001 hour, the effect of enzyme-linked immunosorbent assay (ELISA) on the chickhepatocytes infected for 3 days is comparable to the effect of ELISA on DF-1 cells infected for 7 days. The efficient amplification method established by the invention accelerates the purification process of the subgroup J ALV, thus shortening the time and reducing cost; the method has very good application value in the purification detection of the subgroup J ALV.