63 results about "Pcr cloning" patented technology

The present invention describes the development of a positive selection vector based on regulatory element modulation, wherein such modulation is achieved via insertional reconstruction or destruction of a regulatory element controlling transcription, translation, DNA replication and termination. A positive selection cloning vector pREM5Tc has been developed based on insertional reconstruction of a regulatory element of a reporter gene. The vector pREM5Tc carries the tetracycline resistance reporter gene with no functional -35 region of its promoter, a regulatory element, thus resulting in no expression of the tetracycline resistance gene. Hence a host cell carrying the vector pREM5Tc is unable to produce the tetracycline resistance gene protein resulting in inhibition of its growth in presence of tetracycline. An E. coli consensus -35 region is recognized as 5'-TTGACA-3' and a primer used in polymerase chain reaction (PCR) carries at its 5' end the sequence 5'-TGTCAA-3', which is the complementary sequence of 5'-TTGACA-3'. The PCR-amplified DNA fragment is ligated to pREM5Tc thus reconstructing the functional promoter of the tetracycline resistance reporter gene. Subsequent transformation of a host cell with the recombinant vector (carrying an insert DNA) results in production of the tetracycline resistance reporter gene protein that confers resistance to tetracycline thus allowing only the recombinants to grow in presence of tetracycline. The positive selection vector pREM5Tc greatly reduces, if not eliminates, the number of exonuclease-generated false positive clones.

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.

Reagent kit for predicting 5-hydroxytryptamine re-ingestion inhibitor medicinal effect contains erythrocyte lytic liquid, leukocyte lytic liquid, protein deposit, nucleic acid reserve liquid, PCR reactive mixed liquid with MgC12, dNTP and PCR cloning primer and indicator, DNA polymerase, positive proton, negative control, endonuclease buffer system, limited endonuclease, PCR water, 10X electrophoretic buffer liquid, bromphenol blue 0.25% and sugar solution w / v40%. It extracts gene DNA in biological sampler and has polymerase chain-reaction-limited enzyme fragment polymorphism PCR-RFLP analysis.

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.

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 grass carp hemorrhage antigenic protein, a preparation method and an application thereof, wherein the preparation method comprises the steps: A. according to nucleotide complete sequence of a grass carp reovlrus VP6 gene, designing a primer to perform reverse transcription-polymerase chain reaction; cloning to obtain a VP6 protein gene; B. using EcoRI and SacII to perform enzyme digestion on a product obtained from reverse transcription-polymerase chain reaction in step A and a pPICZB carrier; constructing a pPICZB-VP6 expression carrier; C. electrically transferring recombinant obtained in step B to pichia yeast; coating the pichia yeast on the YPDS flat plate containing ZEOCIN antibiotic, and screening to obtain a high-copy recombinant yeast containing a target gene; and D. obtaining the used antigenic protein by separation and purification. The method is simple and is easy to operate, can be used for carrying out large-scale industrial production by a fermentation tank; the protein is safe and reliable, has immunological competence and can be fully used as the grass carp hemorrhage antigenic protein.

The invention provides a simple, general and effective method for building complex carrier. A randomly designed linker is added on a target fragment 5' in PCR primer design, the target fragment is cloned by PCR, then the PCR cloned target fragment is processed by 3'-5' excision enzyme activity of T4DNA polymerase, a plurality of end-to-end sticky ends are generated, multi-fragment orientation connection, conversion and recon identification are carried out. The method for building complex carrier has high efficiency of rearrangement, connection and conversion and is applicable to building various complex carriers.

The invention discloses a method for detecting the bacterial community structure in rice wine wheat koji by denaturing gradient gel electrophoresis technology. The genomic DNA of the wheat koji sample is extracted by the benzyl chloride method, the bacterial 16S rDNA V3 region is amplified by PCR, and the DGGE electrophoresis band is separated. Secondary PCR and T-cloning after rubber tapping and recovery, selection of positive clones for DGGE band comparison, sequencing after correct comparison, and obtaining relevant microbial information from GenBank. The invention is simple, fast and has good repeatability, and has good guiding significance for analyzing the dominant bacteria and functional microorganisms in rice wine wheat koji.

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 field of plant gene engineering. Specifically, the present invention relates to a PCR cloning soybean PIP (Plasma Intrinsic Protein) gene and an overexpression and interference material by transgenic technology; and also relates to application of the gene to regulate the tolerance of soybean to abiotic adversity stresses thus increasing soybean growth in adversity conditions and improving crop yields. The present invention discloses application of soybean aquaporin gene GmPIP1;2, which is used for constructing transgenic soybean with abiotic stress tolerance. The soybean aquaporin gene PIP1;2 has a nucleoside acid sequence shown as EQ ID NO: 1. The present invention specifically includes transforming soybean cotyledonary node by the nucleotide sequence shown as EQ ID NO: 1, and culturing the transformed soybean cells into transgenic plants.

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 belongs to the technical field of biological fermentation engineering and particularly relates to a method of improving alpha-amylase activity in bacillus subtilis fermentation liquid. A bacillus subtilis strain genome is modified by a gene engineering method, and the method particularly comprises the steps of PCR clonal expansion of sdpBC partial sequence DNA, enzyme digestion and connection, conversion, screening, identification, conversion from homologous single exchange recombinant plasmid to bacillus subtilis 168, detection and identification. According to the method, a bacillus subtilis 168 strain having a relatively clear physiology and conversion background and completing sequencing is taken as a research example, and a sporulation delay protein gene sdpC is subjected to insertion mutation by a homologous recombination method. A preliminary study shows that the viable count and the alpha-amylase activity of the modified strain obtained by the method are better increased and improved, the strain has a better application value, and an application foundation is laid for modification of other strains.

The invention discloses a method for constructing a eukaryote cDNA library and special primers thereof, belonging to the technical field of genetic engineering. The special primers are arranged in two pairs in total, one pair comprises 3RRT and 5RRT in a sequence table, and the other pair comprises 3AP and 5AP in the sequence table. The method comprises the following steps: 1) taking total RNA asa template, and taking the 3RRT and the 5RRT in the sequence table as the primers for synthesizing cDNA of a first chain; 2) using the 3AP and the 5AP in the sequence table for synthesizing the cDNA of double chains; and 3) carrying out agarose gel electrophoresis to remove small-fragment products which are synthesized incompletely, recombining and connecting the cDNA of the double chains with a long-fragment PCR cloning vector of pCR-XL-TOPO, further transforming a connection product to host Escherichia coli XL-Gold competent cells and finally completing the construction of the cDNA library.The method and the special primers can be commonly used for constructing the cDNA libraries for eukaryote samples, wherein mRNAs of the eukaryote samples have the polyA tail ends.

The invention relates to a clone method of SGAE label 3' end cDNA segment, belonging to the field of biotechnology. The method comprises the following steps: reverse transcription is carried out on all mRMAs by utilizing the decorated oligo(dT16), 3-5 cytimidine basic groups are added the 3' end of a first cDNA which is synthesized by murine reverse transcriptase, complementary strand of the first cDNA is synthesized by primers, a pair of primers are synthesized according to the known sequence at the two ends of the cDNA for PCR augmentation, and all the cDNAs are concentrated; the cDNA concentrated in the step one is taken as a template, specific segments containing tag are cloned by adopting semi-nested PCR. By adopting the invention, the specific segments containing tag label can be more easily obtained, the process is simpler and the cost is lower.

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.

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).

A PCR method for preventing the hysteresis pollution by using endodeoxyribonuclease features that the mispairing base is introduced to the terminal 5' of primer for configuring the recognition sequence of endodeoxyribonuclease in the amplified product in such manner that the position of cutting nucleic acid by endonuclease is the 4-20 bases at the upstream of said recognition sequence.

The invention relates to a method for analyzing protein expression difference directly on protein level. The invention has the technical proposals as follows: a fit DNA or RNA random sequence library is constructed, and the cell protein in the DNA or RNA aptamer random sequence library is extracted, so as to obtain a DNA or RNA random sequence library that can reflect protein level change; after subtractive hybridization of target cells and control cells in the DNA or RNA random sequence library that can reflect protein level change is carried out, PCR clone is carried out to eliminate sequence, so as to obtain a differential display library, and sequencing is carried out; the corresponding type of DNA or RNA aptamer is prepared by amplification of marked primer; and the DNA or RNA aptamer is filtered to determine specific proteins having expression difference and function difference. The method has differential display that transfers the expression difference on protein level onto DAN or RAN level by aptamer, thereby achieving the purpose of simply and quickly filtering and expressing differential proteins in high throughput.

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.

Provided are a Cryptomeridiol synthase and a coding gene thereof. Also provided are a Cryptomeridiol synthase and a coding gene, a engineered yeast containing the Cryptomeridiol coding gene, and a use of same in plant breeding and biosynthesis. The cDNA full-length sequence of the Cryptomeridiol synthase gene in Tripterygium wilfordii is obtained by means of polymerase chain reaction cloning. Then, by means of synthetic biology, the engineered yeast containing the Cryptomeridiol synthase gene is constructed to realize the production of Cryptomeridiol in the yeast.

The invention discloses a rice OsGRDP1 protein related to disease resistance of plants and its encoding gene and application. The OsGRDP1 protein is a protein with a sequence 2. The encoding gene of the OsGRDP1 protein is a DNA molecule with a sequence 1. Through TAIL-PCR, a T-DNA insertion site is cloned. Through insertion of T-DNA, the up-regulated expression of the OsGRDP1 gene is caused. Through overexpression of the OsGRDP1 gene in the wild-type rice, it is proved that the overexpression of the OsGRDP1 gene can cause spontaneous rice lesion mimic and can improve resistance to rice blast and bacterial blight. The OsGRDP1 gene has a good application prospect of improving crop immunity. Through the gene engineering, the expression level of the OsGRDP1 gene is adjusted and controlled to be used for molecular breeding and has a potential application value in improving crop disease resistance.

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.

A method for constructing Saussurea involucrate cDNA library, screening encoding gene of Saussurea involucrate by degenerate primer and cloning chs by specific primer, and its chs full-length cDNA sequence and encoded amino-acid polypeptide sequence are disclosed. The length of cDNA is 1,313bp and it encodes 389 amino-acid polypeptides, it can be used to construct positive-sense gene constructor, control plant or plant culture and improve flavone yield.

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.

The invention provides a novel efficient selectable marker-free corn transgenic vector system construction method and an application thereof. By a PCR cloning technology, related fragments of Mu transposons are obtained from highly active Mu lines of corn; a binary plasmid vector pCAMBIA1300 is used as a skeleton to respectively construct novel efficient binary plasmid vectors pMuE and pMuDR; and through an agrobacterium-mediated genetic transformation method, Luyuan 92 selfing line of corn is transformed, and a selectable marker-free transgenic plant is screened out from progeny. According to the invention, transformation efficiency of transgene can be enhanced. By the vector system, a marker-free bio-safety transformant can be directly obtained in future. The vector system especially can provide a reliable and effective tool for marker-free gene transformation of important grain crops such as corn, rice and the like. The method provided by the invention has important theoretical significance and application value.

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.