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 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 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 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 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 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.

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 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.