63 results about "Pcr cloning" patented technology

The present invention describes a methodology for generating high fidelity PCR products, and also cloning of such high fidelity PCR products in a suitable vector. Generation of polymerase-induced mutant fraction of target sequences during PCR amplification is linearly proportional to the number of doublings of the target sequences. Thus the high fidelity PCR products are generated by minimizing the number of doublings of the target nucleic acid sequences during PCR amplification. Minimization of number of doublings of the target sequences is achieved by reducing the number of cycles of PCR amplification of the target sequences. The high fidelity PCR products thus obtained are then cloned into a suitable vector. As an example, a 960 bp target sequence from E. coli DNA was PCR-amplified only for 3 cycles, and it was then directly cloned into a positive selection cloning vector pRGR2Ap. The functional analysis of the inserts in all clones showed that the clones carried functionally wild-type DNA fragments, and hence the inserts most probably carry no mutation. Cloning of PCR products obtained from 3 cycles of amplification, instead of 30 cycles of amplification, theoretically achieves 10-fold reduction of mutations in the cloned fragments. The invention also contemplates cloning of a target cDNA obtained by primer extension.

The present invention relates to a beta-seminase MANB48, its coding gene and application. It is characterized by that said invention uses genome DNA of bacillus circulans as template, and utilizes reverse PCR clone to obtain beta-seminase gene. The total length of said gene is 2079bp, said gene codes a mature beta-seminase containing 372 amino acids and a signal peptide containing 31 amino acids. The invented beta-seminase has good heat resistance and capability of resisting trypsase, and can be used as a feed additive.

The present invention relates to process of fast extracting AM fungal genome DNA in plant rhizosphere soil environment, and belongs to the field of molecular biology and applied microbiology. The process includes the first wet screening to eliminate great amount of PCR proliferation limiting factors from rhizosphere soil, collecting AM fungal related structures of rhizosphere soil for DNA extraction, mechanically breaking wall and adding CTAB to promote DNA release, and further purifying with Chelex-100 resin. The process is easy, simple and fast, and has great DNA extracting amount, capacity of being concentrated and purified and other advantages. The present invention has excellent application foreground.

The invention relates to a chlamydia pneumonia genetic engineering expression artificial antigen, and a method for preparing the antigen. The method comprises the steps of: cloning and amplifying a chlamydia pneumonia MOMP (major outer membrane protein) antigen gene sequence by PCR (polymerase chain reaction), building a prokaryotic expression vector, expressing chlamydia pneumonia MOMP antigen protein by escherichia coli, and obtaining a reconstructed chlamydia pneumonia MOMP antigen with a three-dimensional structure and immunological competence by a dialysis method, a gradient dilution method and a gel chromatography renaturation inclusion body. A fast detection method for detecting the chlamydia pneumonia antigen antibody is provided. The method comprises the application of the chlamydia pneumonia antigen; and the invention provides a fast detection reagent for detecting the chlamydia pneumonia antibody. The reagent contains the chlamydia pneumonia antigen. According to the invention, the chlamydia pneumonia antigen is provided. The antigen has high specificity. The method for preparing the antigen, the method for fast measuring the chlamydia pneumonia antibody, and the reagent for fast measuring the chlamydia pneumonia antibody are provided by the invention.

The invention belongs to the technical field of shelled animal gene engineering, and discloses a novel penaeus monodon ALFpm12 antibacterial peptide, and a preparation method and applications thereof.The novel penaeus monodon ALFpm12 antibacterial peptide is composed of 123 amino acids, the molecular weight is 13.54kDa, and the signal peptide is composed of 25 amino acids. The highest homology ofALFpm12 with disclosed penaeus monodon ALF antibacterial peptide is only 50%, up-regulating in organs of penaeus monodon infected by vibrio parahaemolyticus is realized, and the expression peak valueis achieved in 4h; the gene sequence can be obtained through RT-PCR of total RNA extracted from penaeus monodon tissues. The novel penaeus monodon ALFpm12 antibacterial peptide possesses inhibiting and killing effect on Staphylococcus aureus and Exiguobacterium Collins; the sterilization efficiency is high; the using amount is low; effect lasts long; production is simple; bacteria in sea water can be killed; and the application prospect in the field of aquaculture is promising.

The invention belongs to the technical field of crustacean genetic engineering, and discloses a Ppenaeus monodonantibacterial peptide ALFpm10 and a preparation method thereof. The Ppenaeus monodonantibacterial peptide ALFpm10 is prepared from 132 amino acids, the molecular weight is 15.3 kDa, and the signal peptide is prepared from 27 amino acids; the full-length cDNA of the ALFpm10 antibacterialpeptide is prepared from 517 bases, and obtained from RT-PCR cloning in the total RNA of a Penaeus monodon blood cell; the ALFpm10 antibacterial peptide can be obtained by fusion expression with SUMOprotein through a prokaryotic expression system, which is a solid phase chemosynthetic method. The ALFpm10 antibacterial peptide disclosed by the invention has antibacterial and bactericidal effects against Staphylococcus aureus, coastal Exiguobacterium,algae-occupying Bacillusand the like, and has the characteristics of high bactericidal efficiency, small usage amount, long lasting, and capable of suppressing seawater bacteria, and shows a broad application prospect in the aquaculture industry.

The invention discloses a single domain antibody resisting to human nerve dynein 1 and a preparation method of the single domain antibody and belongs to the technical field of genetic engineering and antibody engineering. The preparation method comprises the steps of cloning genes of the single domain antibody resisting to the human nerve dynein 1, introducing the genes into a vector, and constructing a recombinant vector; introducing the recombinant vector into a host bacterium, and constructing a recombinant bacterium which expresses the single domain antibody resisting to the human nerve dynein 1; fermenting and culturing the recombinant bacterium, separating and purifying fermentation liquor, and obtaining the single domain antibody resisting to the human nerve dynein 1. According to the single domain antibody resisting to the human nerve dynein 1 and the preparation method of the single domain antibody, a hybridoma cell strain A6 of the monoclonal antibody antibody resisting to the human nerve dynein 1 serves as the template, and RT-PCR cloning is conducted, so that the heavy chain variable region gene (V<H>) of the antibody is obtained; the gene is introduced into the plasmid vector pET-22b (+), and the recombinant vector pET-22b(+)-V<H> is constructed; the recombinant vector is introduced into the expression strain E. coli BL21(DE3), IPTG induces expression of the single domain antibody resisting to the human nerve dynein 1, and then purification and identification are conducted.

A method for selectively obtaining a natural variant of an enzyme having activity includes (1) a step of detecting an ORF sequence of a protein having enzyme activity from a genome database including base sequences of metagenomic DNA of environmental microbiota; (2) a step of obtaining at least one PCR clone including the ORF sequence having a full length, a partial sequence of the ORF sequence, or a base sequence encoding an amino acid sequence which is formed by deletion, substitution, or addition of at least one amino acid residue in an amino acid sequence encoded by the ORF sequence, by performing PCR cloning on at least one metagenomic DNA of the environmental microbiota by using a primer designed based on the ORF sequence; (3) a step of determining a base sequence and an amino acid sequence which is encoded by the base sequence for each PCR clone obtained in the step (2); and (4) a step of selecting a natural variant of an enzyme having activity by measuring enzyme activity of proteins encoded by each PCR clone obtained in the step (2).

The invention discloses a brassica napus BnGLABRA2 promoter and a preparation method and application thereof. The method comprises the following steps of: A, cloning a promoter sequence by genomic walking, namely extracting genomic DNA (Deoxyribose Nucleic Acid) by using an SDS (Sodium Dodecyl Sulfonate) cracking method, performing genomic walking, and amplifying PCR (Polymerase Chain Reaction) cloned rape twice during each genomic walking; B, performing bioinformatics analysis on the promoter sequence, namely performing homology comparison on a cloned sequence by BLASTN, wherein a promoter sequence and an upstream promoter element are obtained from a promoter core element and an upstream cis acting element by using promoter prediction software; and C, amplifying a full-length promoter and a missing promoter and constructing a pMD18-T-GP vector, namely designing and amplifying a primer of each missing segment by taking the genomic DNA as a template according to the previously obtained promoter sequence for PCR amplification. The application of the brassica napus BnGL2 genomic full-length promoter SEQ ID NO:1 to leaf epidermal hairs, seeds, root parts, entire veins and stele leaf veins is disclosed. The method has the advantages of easiness in operating, stable and reliable result. The brassica napus BnGLABRA2 promoter has biological safety and development and application values.

The invention relates to an insecticidal crystalline gene crylAc22 which is separated and cloned from bacillus thuringiensis (Bt) wild strains W015-1 and has high efficient insecticidal activity for lepidoptera insect, and application thereof. The invention adopts a reverse PCR clone method for cloning CrylAc full length gene from Bt strains w015-1, an open reading frame (ORF) of the gene is 3534bp, protein consisting of 1178 amino acids is coded, the whole protein contains three endotoxin protein conserved domains, and is named CrylAc22 by international Bt insecticidal crystalline gene name committee, and GenBank registration sequence number is EU282379. By using engineering bacteria or purified expression protein, the invention has high-active poisoning effect for lepidoptera insect Plutella xylostella.

The invention relates to heterogeny expression and purification method of a human RANTES protein with chemotaxis. A Pichia pastoris expression system with the advantages of high expression, high stability, high exudation and low cost is adopted and the protocols in molecular biology and the protein expressing and purifying technology which include cloning a human RANTES gene, building a human RANTES fusion protein expression vector and the methanol induction expressing and purifying authentication of the human RANTES fusion protein through PT-PCR, and the like are adopted for realizing the exudation expression of the human RANTES protein. In order to be convenient for the separation and purification of the human RANTES protein, a 6 multiplied by His purifying label is fused at the N end of a target protein, and moreover, an enterokinase(EK) restriction enzyme locus is added between His-tag and the RANTES protein for building the RANTES fusion protein; then the human RANTES protein with higher purity and larger yield is obtained through affinity chromatography and EK restriction enzyme locus; and the protein can be used for preparing the medicaments for curing multiple sclerosis arthritis, rheumatoid arthritis, organ transplantation rejection, cardiovascular and cerebrovascular disease, knub and HIV.

The coat protein of lettuce big-vein virus (LBVV) was purified from highly purified LBVV, and its partial amino acid sequences were determined. An RNA encoding the coat protein of LBVV was cloned by polymerase chain reaction using primers designed based on the determined amino acid sequences information, and its primary structure was elucidated. Moreover, the present inventors succeeded not only in isolating RNA molecules of a plurality of LBVV-encoded proteins, including LBVV polymerase, by carrying out 3′RACE and 5′RACE using primers designed based on the resulting sequence information, but also in determining their primary structure. It was found that the use of these made it possible to produce lettuce resistant to LBVV and to diagnose infections with LBVV.

An SOS gene has been isolated through PCR cloning from Thellungiella halophila and is designated TSOS1. The polynucleotide encodes a transmembrane protein with similarities to plasma membrane Na+ / H+ antiporters from bacteria and fungi. The invention encompasses the TSOS1 gene, the corresponding protein, closely related polypeptides that confer salt tolerance, and related polynucleotides that encode a polypeptide that has Na+ / H+ antiporter activity.