32 results about "Cdna cloning" patented technology

The invention relates to a virus antibody research and provides a method for constructing a hog-cholera virus infectious cDNA carrier having a molecule mark. The method comprises the following steps of: (1) diluting a hog-cholera live vaccine to 1 portion per milliliter as the experimental material, after extracting the head RNA, amplifying 6 corresponding cDNA fragments through RT-PCR section according to designed 6 pairs of primers, (2) leading in the corresponding molecule marks from F1 and F5 of cDNA fragments, (3) reforming plasmids required by overall length cDNA cloning, and (4) constructing the overall length cDNA carrier. A hog-cholera virus lapinized vaccine C stem infectious cDNA carrier pA-FL22 having a molecule mark can be acquired through the method of the invention. The permissive cells of RNA electro transferred hog-cholera virus acquired by the carrier can save the infectious progeny virus which can stably inherit the molecule mark led in the construction. By using the saving technique system of the infectious cDNA carrier and the progeny virus, the copy stem and the pathopoiesia reason of the virus can be deeply researched and the foundation for developing new marked vaccines can be settled.

The present invention provides a method of cDNA sequencing by circulating subtraction, which comprises the steps of: (1) constructing the cDNA original libraries with high quality using the organism tissues as materials; (2) homogenizing the cDNA original libraries of step (1) according to the graded C0t value, thereby obtaining the homogenized cDNA libraries corresponding to the graded C0t value; (3) selecting and sequencing the clones from each of the homogenized cDNA libraries of the preceding step; (4) hybridizing and subtracting the homogenized cDNA libraries of the preceding step by using the sequenced cDNA clones; (5) repeating steps (3) and (4) 1-5,000 times. Further, a step of preliminary subtractive hybridization may be included in the method. By using said method, one can sequence cDNA of certain tissues or species efficiently and comprehensively.

The invention provides a building method of a transposable element carrier expressing pig telomerase reverse transcriptase, and application in building a pig immortalization cell line, and belongs to the field of biotechnology research. The building method includes the steps of cloning of a promoter of a pig protein translation elongation factor 1a and detection of transcriptional activity, building of an SB transposable element expression vector and exogenous gene integration, pig telomerase reverse transcriptase cDNA cloning, activity detection and building of the pig fibroblast immortalization cell line. Compared with other methods, the pig immortalization cell line built through the transposable element carrier comprises non-transformed normal cells, does not contain resistance genes, virogenes or oncogenes, and is suitable for cell physiological function researching, pig virus separation cultivation and vaccine production.

The invention relates to a construction method of a Membrane protein cDNA library and an application thereof. The method constructs a eukaryotic expression vector used for the specificity selection of the membrane protein. The expression vector can perform clone recombination with any cDNA clone library provided with the recombination site attL of Lambada bacteriophage so as to acquire a cDNA expression library. After the transfection of COS-1 cell, expression membrane protein clone is further selected by immunofluorescence so as to acquire the membrane protein cDNA expression library. As the application of the method, the liver lymphocyte membrane protein cDNA library of a C57BL / 6 rat is built up. The method is proved to be an effective proposal of the high flux specificity selection membrane protein through the identification and analysis of the library, and the method provides a powerful method to describe the map of the membrane protein of the cell surface of certain issue and to research the unknown membrane protein.

The invention discloses a porcine reproductive and respiratory syndrome virus with a preservation number of CCTCC NO:V 202020. Accordingly, the inventor constructs virus genome full-length infectiousclone pGXAM and establishes a corresponding construction method and a gene insertion method. According to a vector, Gxnn1396-P96 is used as a female parent, and Gxnn1396-P96 is used as a male parent,a PRRSV full-length infectious clone vector is constructed by means of molecular biology, an independent transcription regulating sequence (TRS) is inserted into ORF4 and ORF5a on the basis of cDNA cloning, and the PRRSV recombinant virus for expressing a green fluorescent protein (GFP) gene is constructed. The successful construction of the fluorescence labeled virus lays a foundation for the development of recombinant porcine reproductive and respiratory syndrome virus genetic engineering vaccines.

The invention relates to a cDNA nucleotide sequence which encodes S6K1 protein and is separated from a goat muscle cell and the corresponding amino acid sequence. The invention finds a conservation region by comparing the cDNA nucleotide sequences of the known human, rat, rabbit and cattle S6K1 genes and then designs a pair of primers for a cDNA coding area fragment of an RT-PCR amplified goat S6K1 gene according to the cDNA sequence of a cattle S6K1 gene (GenBank landing number is AY396564), the primers are used for amplifying the specific fragment by the PT-PCR method, the nucleotide sequence of 1563bp of the cDNA coding area of the goat S6K1 gene and the corresponding amino acid sequence are obtained after sequencing. The obtained 1563bp nucleotide sequence can be further used for full-length cDNA cloning of the goat S6K1 gene and detecting the tissue expression specificity of the S6K1 gene by the RT-PCR or the hybridization method, which can also be used for preparing an antibody for detecting the S6K1 after recombinant expression.

The present invention provides a rabies virus CTN strain whole genome cDNA infectious clone, as well as a method of using reverse genetics technology to save the active rabies virus. The present inventive method includes: using the single restriction enzyme sites in the CTN strain of rabies virus full-length genome to divide the full-length virus into four segments to amplify, simultaneously using the green fluorescent protein gene to substitute 423bp base at the pseudo-gene point, cloning in the pVAX-R expression vector, to construct a recombinant full-length plasmid of the virus genome; performing PCR amplification of nucleoprotein, phosphoprotein, glycoprotein and transcription large protein gene sequence of the CTN strain of the rabies virus, and cloning the amplified fragment into a vector pVAX1, to constitute four auxiliary plasmids in the reverse heredity system; and then using the five plasmids to jointly transfect cells, to rescue the rabies virus. The present invention successfully saves the rabies virus, and has a great significance in the study of the pathogenic mechanism of rabies virus, development of new vaccines, viral vectors and the like areas.

The present invention describes the whole nucleotide sequence of beet black scorch virus (BBSV). The cDNA cloning of BBSV whole genome may be transcribed under the control of eukaryotic and prokaryotic promoter to produce viral RNA with infection capacity to host plant. Taking the insertion of medusa green fluorescent protein gene as example, the present invention shows that obtained recombinant BBSV may be used as carrier for carrying virus to express exotic gene in plant.

The invention discloses a method for synthesizing double-stranded RNA used for inhibiting the growth of Chilo suppressalis and using acy1-CoA desaturase SexiVPAE genes, which comprises the following steps of: designing a corresponding PCR primer according to the cDNA cloning sequence of acy1-CoA desaturase SexiVPAE genes of Chilo suppressalis; amplifying the cDNA fragments of acy1-CoA desaturase SexiVPAE genes Through a PCR method, and carrying out in-vitro transcription on the obtained object so as to synthesize dsRNA; and adding the dsRNA into a Chilo suppressalis artificial feed, and feeding Chilo suppressalis with the artificial feed containing the dsRNA. Artificial feeding studies show that high-concentration dsRNA can significantly increase the death rate of Chilo suppressalis larvae and inhibit the growth of the larvae, and the results lay the important foundation for effectively controlling bivoltine by further using the dsRNA sequence in production.

The invention discloses a porcine reproductive and respiratory syndrome virus, and the collection number of the porcine reproductive and respiratory syndrome virus is CCTCC NO: V 202020. Thereby, inventors construct a virus genome full-length infectious clone pGXAM and establish a corresponding construction method and a gene insertion method. The vector takes GXNN1396-P96 as a female parent, and a PRRSV full-length infectious cloning vector is constructed by means of molecular biology, and on the basis of cDNA cloning, an independent transcriptional regulatory sequence (TRS) is inserted into ORF4 and ORF5a, and accordingly, a PRRSV recombinant virus for expressing a green fluorescent protein (GFP) gene is constructed. Successful construction of the fluorescence labeled virus lays a foundation for development of recombinant porcine reproductive and respiratory syndrome virus genetic engineering vaccines.

The invention discloses a canine distemper virus CDV-3 strain infectious cDNA cloning and constructing method and application thereof. A canine distemper virus CDV-3 strain is used as a template, theoverall length of the CDV-3 is divided into 5 fragments, and RT-PCR amplification is performed. Through enzyme-cutting splicing, the 5 fragments are sequentially inserted into a eukaryotic vector, besides, a hammerhead ribozyme sequence and a hepatitis D ribozyme sequence are respectively added to an F1 head end and an F5 tail end, and canine distemper virus CDV-3 strain infectious cDNA clone is obtained; then three auxiliary plasmids expressing CDV-3N, P and L proteins are constructed; the canine distemper virus CDV-3 infectious cDNA clone and the three auxiliary plasmids are used for performing cotransfection on 293T cells so that saved recombinant CDV-3 viruses (rCDV-3) are obtained. Through research, the highest titer of the obtained rCDV-3 virus can reach 107.667TCID50 / mL, which is 10times higher than that of wtCDV-3. After Vero cells are infected with the rCDV-3, within 36h after infection, the highest virus titer can be achieved; but after the Vero cells are infected with the wtCDV-3, within 72h after infection, the virus content can reach the highest value. A base is established for developing a canine distemper virus novel vaccine and studying the infective mechanism through the canine distemper virus CDV-3 strain infectious cDNA cloning and constructing method disclosed by the invention.

The invention discloses RNA compatible medium and its manufacturing method used to separate and identify RNA singleness conjugated protein. It includes the following steps: fixing one end of DNA fragment of the plasmid pSP65 on glass dust of amino silane to form double chain DNA-glass dust; cloning target RNA gene or cDNA into another reciprocal clone site plasmid Psp64 to gain recombinant plasmid; gaining DNA fragment with extended nucleotide sequence by enzyme cutting; transcript RNA target sequence with RNA complementation fragment which is complemented with the plasmid pSP65 DNA fragment; heating the double chain DNA-glass dust and RNA target sequence together then cooling to make the RNA target sequence and signal chain DNA hybridize to form new RNA compatible medium which has the advantage of easy preparation, strong selectivity, repeatable utilization, etc.

The invention discloses cDNA cloning of an epidemic encephalitis B / yellow fever chimeric virus. The nucleotide sequence of the virus is as shown in SEQ ID NO.1. The invention further discloses the epidemic encephalitis B / yellow fever chimeric virus, application thereof and a vaccine preventing yellow fever. The chimeric virus Chimeri-JYF is small in toxicity and good in immune protection function. The vaccine prepared through the virus can effectively prevent yellow fever virus infection, is high in safety, can be used for replacing existing yellow fever live attenuated vaccines (17D strains), effectively improves clinical use safety while the immune protection function is guaranteed, and is good in application prospect.

Disclosed is a method for producing a cDNA library having a reduced content of a cDNA clone derived from a highly expressed gene with high efficiency. In a method for producing a cDNA library by using a double-stranded DNA primer containing an oligo-dT and also using mRNA as a template, the content of a cDNA clone derived from a highly expressed gene in the cDNA library can be reduced by allowing a probe to coexist, wherein the probe has such a property that the probe can bind to mRNA of a highly expressed target gene to inhibit the occurrence of a cDNA extension reaction with a reverse transcriptase.

The invention discloses a special primer capable of detecting different strains of potato viruses and a design method thereof. The special primer provided by the invention comprises at least one of a primer pair I, a primer pair II, a primer pair III, a primer pair IV, a primer pair V and a primer pair VI in a sequence table. The invention also provides a method and condition for detecting the potato viruses by using the special primer. The invention also provides a design method of the special primer. RC-PCR (Rapid cDNA Cloning- Polymerase Chain Reaction) detection based on the special primer has high specificity, high sensitivity, high detection speed, simplicity in sample pretreatment and high repeatability, is easy and convenient to operate and is easy to analyze; the special primer screened by means of the design provided by the invention has 100 percent of similarity to the potato viruses, and can be used for detecting different strains of potato viruses which are reported or found; and the special primer has a wide application value in the detection of potato viruses.

The invention provides an A-type Seneca virus SVA / HN / 11 / 2017 Lanzhou Veterinary Institute of Chinese Academy of Agricultural Sciences full-length infectious cDNA clone as well as a construction methodand application thereof, and belongs to the technical field of viruses. On the basis of carrying out whole genome determination on SVA / HN / 11 / 2017 Lanzhou Veterinary Institute of Chinese Academy of Agricultural Sciences, a T7 promoter sequence is introduced to the 5' end of a genome cDNA sequence, a Not I restriction enzyme cutting site sequence is introduced to the 3' end, meanwhile, an Xho I restriction enzyme cutting site is introduced to a 2B gene, the Xma I restriction enzyme cutting site is eliminated, and recombinant plasmids containing SVA / HN / 11 / 2017 Lanzhou Veterinary Institute of Chinese Academy of Agricultural Sciences full-length cDNA are constructed. The invention further discloses application of the virus obtained on the basis of infectious cDNA cloning rescue in preparationof an A-type Seneca virus labeled vaccine. The infectious cDNA clone and the preparation method provided by the invention provide an effective platform for deep development of basis and application research of SVA, and have important scientific application value.

The invention provides a recombinant coxsackie B3 virus with a fluorescent protein tag, the virus is an infectious recombinant virus rCV-CS and a recombinant virus rCV-GFP for expressing GFP, and the sequence is as shown in SEQ ID NO: 1. The construction method comprises the following steps: on the basis of a CVB3 full-length infectious cDNA cloning plasmid pCV, constructing a CVB3 full-length infectious cloning plasmid pCV-CS containing multiple cloning sites MCS, and adding a 2Apro enzyme digestion recognition sequence in front of the MCS; amplifying a GFP fragment by taking the pEGFP as a template to obtain a GFP coding gene; the method comprises the following steps: introducing a GFP coding gene into multiple cloning sites of pCV-CS, and constructing a CVB3 whole-genome infectious cloning plasmid pCV-GFP for expressing the GFP; and transfecting the pCV-GFP through a liposome, and amplifying the pCV-GFP to obtain the recombinant coxsackie B3 virus rCV-GFP with a fluorescent protein tag. The technical scheme of the invention is used for preparing tracer viruses in living animal cells, and a remarkable effect is generated.

The present invention provides a rabies virus CTN strain whole genome cDNA infectious clone, as well as a method of using reverse genetics technology to save the active rabies virus. The present inventive method includes: using the single restriction enzyme sites in the CTN strain of rabies virus full-length genome to divide the full-length virus into four segments to amplify, simultaneously using the green fluorescent protein gene to substitute 423bp base at the pseudo-gene point, cloning in the pVAX-R expression vector, to construct a recombinant full-length plasmid of the virus genome; performing PCR amplification of nucleoprotein, phosphoprotein, glycoprotein and transcription large protein gene sequence of the CTN strain of the rabies virus, and cloning the amplified fragment into a vector pVAX1, to constitute four auxiliary plasmids in the reverse heredity system; and then using the five plasmids to jointly transfect cells, to rescue the rabies virus. The present invention successfully saves the rabies virus, and has a great significance in the study of the pathogenic mechanism of rabies virus, development of new vaccines, viral vectors and the like areas.

The invention discloses a PRSV-W attenuated vaccine and application thereof. Mutation is introduced into a HC-Pro gene of PRSV-W through a site-directed mutation technology in a site-directed mode onthe basis of PRSV-W full-length infectivity cDNA cloning, and an amino acid site for controlling virus virulence is explicited, so that a PRSV-W attenuated mutant with remarkably reduced virulence isobtained. The research finds that attenuated vaccines K125D, G317K and N137A have good crossed protection effect on the PRSV-W high virulence strain, so that melon and pumpkin plants are prevented from being infected by the high virulence strain.

A number of cDNA clones whose products may interact with D1 receptors in vivo were identified. One of the clones, P24, was characterized further. P24 is localized in dendrites and spines of pyramidal cells in PFC. The extent of overlap between P24 expressing and D1 receptor expressing pyramidal cells appeared to be 100%. In contrast, only a limited number D1 receptor antibody labeled neurons in caudate expressed P24. P24 lowers the threshold of D1 receptor response to dopamine (DA) by an order of magnitude. Sequence similarity suggests P24 is a diverged member of the RAMP family. The P24 protein is therefore referred to as a D1 DA RAMP, calcyon. The isolated protein and nucleotide molecule encoding the protein, as well as primers for the nucleotide, are described. The protein and compounds modifying DA binding to the receptor or calcium release which is mediated by the Calcyon, are useful in research studies, drug screening, and therapeutically.

A recombinant canine TRPV2 channel which has been prepared by cDNA cloning and polymerase chain reaction techniques is disclosed. Expression systems for these channels and an assay using the expression systems are also disclosed. The recombinant TRPV2 channel can be used in assays to evaluate compounds which directly or indirectly interact with or bind to TRPV2 channel.

The invention relates to an SARS-CoV-2 full-length cDNA cloning single-copy plasmid and a construction method thereof. Specifically, In-Fusion and Gibson Assembly seamless cloning are combined to perform full-length cDNA synthesis and cloning on an SARS-CoV-2 genome. A single-copy plasmid pBac-mini (named pY1B In the early stage and pBacm In the present stage) which is based on a BAC vector and is stable In replication and smaller In molecular weight is constructed, SARS-CoV-2 full-length cDNA is cloned to the vector, a recombinant single-copy plasmid pBacm-SARS-CoV-2 is constructed, and a Recombineering technology is utilized to perform beneficial base modification, gene knockout and other genetic engineering modification on the full-length cDNA.

A recombinant canine TRPV2 channel which has been prepared by cDNA cloning and polymerase chain reaction techniques is disclosed. Expression systems for these channels and an assay using the expression systems are also disclosed. The recombinant TRPV2 channel can be used in assays to evaluate compounds which directly or indirectly interact with or bind to TRPV2 channel.

The invention discloses RNA compatible medium and its manufacturing method used to separate and identify RNA singleness conjugated protein. It includes the following steps: fixing one end of DNA fragment of the plasmid pSP65 on glass dust of amino silane to form double chain DNA-glass dust; cloning target RNA gene or cDNA into another reciprocal clone site plasmid Psp64 to gain recombinant plasmid; gaining DNA fragment with extended nucleotide sequence by enzyme cutting; transcript RNA target sequence with RNA complementation fragment which is complemented with the plasmid pSP65 DNA fragment;heating the double chain DNA-glass dust and RNA target sequence together then cooling to make the RNA target sequence and signal chain DNA hybridize to form new RNA compatible medium which has the advantage of easy preparation, strong selectivity, repeatable utilization, etc.

The invention relates to a cDNA which encodes mTOR protein and is separated from a goat testicle cell and the corresponding amino acid sequence. The invention finds a conservation region by comparing the nucleotide sequences of the known human, rats and mice mTOR genes and then designs a pair of degenerate primers for amplifying the 3' end fragment of a cDNA coding area of a goat mTOR gene by RT-PCR, the primers are used for amplifying the specific fragment by the PT-PCR method, the nucleotide sequence of 612bp of the 3' end of the cDNA coding area of the goat mTOR gene and the corresponding amino acid sequence are obtained after sequencing. The obtained 612bp nucleotide sequence can be further used for full-length cDNA cloning of the goat mTOR gene and detecting the tissue expression specificity of the mTOR gene by the RT-PCR or the hybridization method, which can also be used for preparing an antibody for detecting the mTOR after recombinant expression.

The invention relates to a construction method of a Membrane protein cDNA library and an application thereof. The method constructs a eukaryotic expression vector used for the specificity selection of the membrane protein. The expression vector can perform clone recombination with any cDNA clone library provided with the recombination site attL of Lambada bacteriophage so as to acquire a cDNA expression library. After the transfection of COS-1 cell, expression membrane protein clone is further selected by immunofluorescence so as to acquire the membrane protein cDNA expression library. As theapplication of the method, the liver lymphocyte membrane protein cDNA library of a C57BL / 6 rat is built up. The method is proved to be an effective proposal of the high flux specificity selection membrane protein through the identification and analysis of the library, and the method provides a powerful method to describe the map of the membrane protein of the cell surface of certain issue and to research the unknown membrane protein.

A method for efficiently constructing a cDNA library having a reduced content of cDNA clones derived from a highly expressed gene is provided. According to the method for constructing a cDNA library using a double-stranded DNA primer having oligo dT and mRNA as a template, the proportion of cDNA clones derived from a highly expressed gene in a cDNA library was decreased through coexistence with a probe having a property of binding to the mRNA of the highly expressed target gene so as to inhibit a cDNA extension reaction resulting from reverse transcriptase.