39 results about "Double mutation" patented technology

The invention is based on the reaction of Duplex Mutational Vector in a cell-free system containing a cytoplasmic cell extract and a test plasmid. The reaction specifically converts a mutant kanr gene to recover the resistant phenotype in transformed MutS, RecA deficient bacteria. Using this system a type of Duplex Mutational Vector termed a Non-Chimeric Mutational Vector, having no RNA:DNA hybrid-duplex is shown to be an effective substrate for eukaryotic enzymes. The invention concerns the use of Non-Chimeric Mutational Vectors protected from 3' exonuclease attack in eukaryotic cells. Such protection can be conferred by replacement of a tetrathymidine linker by a nuclease resistant oligonucleotide, such as tetra-2'-O-methyl-uridine, to link the two strands of the recombinagenic oligonucleobase.

The invention is based on the reaction of recombinagenic oligonucleotides in a cell-free system containing a cytoplasmic cell extract and a test duplex DNA on a plasmid. The reaction specifically converts a mutant kanr gene to recover the resistant phenotype in transformed MutS, RecA deficient bacteria and allows for the rapid and quantitative comparison of recombinagenic oligonucleobases. Using this system a type of Duplex Mutational Vector termed a Heteroduplex Mutational Vector, was shown to be more active in than the types of mutational vectors heretofore tested. Further improvements in activity were obtained by replacement of a tetrathymidine linker by a nuclease resistant oligonucleotide, such as tetra-2'-O-methyl-uridine, to link the two strands of the Duplex Mutational Vector and removal of the DNA-containing intervening segment. The claims concern Duplex Mutational Vectors that contain the above improvements. In an alternative embodiment the claims concern a reaction mixture containing a recombinagenic oligonucleobase, a cell-free enzyme mixture and a duplex DNA containing a target sequence. In yet an alternative embodiment, the invention concerns the use of such mixture to test improvements in recombinagenic oligonucleobases, as well as to test the effects of compounds on the activity of the cell-free enzyme mixture and also to make specific changes in the target DNA sequence.

The present invention provides RecA mutant proteins, having either a single mutation or a double mutation. The RecA mutant proteins are highly proficient in both SSB displacement and steady state binding of DNA in the presence or absence of SSB as compared to the wild-type protein. The single RecA mutant, RecAΔC17, has 17 amino acid residues removed from the carboxyl terminus. The double mutant RecA, RecAΔC17 / E38K, combines the 17 amino acid residue C-terminal deletion of RecAΔC17, with a single amino acid change from Glutamate to Lysine at position 38. These RecA mutant proteins are pH sensitive allowing control over formation of products. Hence, methods of using the novel RecA mutants and kits having the RecA mutants as components thereof are also contemplated by the present invention.

Modified polypeptides based on the botulinum neurotoxin A heavy chain containing the double mutation Trp-Tyr->Leu-Ser in the ganglioside binding motif Ser-X-Trp-Tyr do not bind polysialogangliosides and nerve endings. The polypeptides are useful in the preparation of nontoxic vaccines against the effects of C. botulinum infection. The modified polypeptides are also useful as vehicles for the transepithelial delivery of diagnostic and therapeutic entities, through formation of conjugates between the polypeptides and the diagnostic or therapeutic entities.

The invention discloses a nitrilase mutant and its application in R-o-chloromandelic acid synthesis. The mutant is obtained by single mutation or double mutation of the 132nd threonine or 189th phenylalanine on the amino acid sequence shown as SEQ ID NO.2. Compared with non-mutant nitrilase, the enzyme activity is increased by 3.70 times and reaches 1.08U / mg, and the enantioselectivity is up to 99%. Also, the result shows that in a two-phase system (with ratio of toluene to water being 2:8) the nitrilase mutant can catalyze 300mM o-chloromandelonitrile, and the yield is 90.8%. Through fed batch of the substrate, a maximum of 500mM o-chloromandelonitrile can be catalyzed, the yield is 90%, and the enantioselectivity is greater than 99%.

The present invention pertains to a Salmonella microorganism having an attenuating mutation which disrupts the expression of a gene located within the Spi2 pathogenicity island, and an auxotrophic mutation. The microorganism therefore has a double mutation which helps prevent reactivity of the microorganism while maintaining the effectiveness of the microorganism to elicit an immune response. The present invention also pertains to vaccine compositions and methods for treating and preventing a Salmonella infection in a patient.

The invention discloses a mutant for trehalose synthase as well as a preparation method and an application of the mutant and belongs to the fields of genetic engineering and enzyme engineering. The mutant is prepared by the following steps: mutating 289th bp glutamate near the active center of the trehalose synthase of Thermobifida fusca YX into glycine to obtain a mutant E289G; mutating 295th bp histidine into an asparagine mutant H295N; mutating 344th bp methionine into a lysine mutant M344K; and mutating 367th bp methionine into a leucine mutant M367L, and carrying out double mutations on the basis of the H295N to obtain mutants H295N / E289G, H295N / M344K, H295N / M367L and H295N / M344K / M367L. According to the mutant disclosed by the invention, although a substrate contains a certain quantity of glucose, the transformation efficiency of preparing trehalose by virtue of the trehalose synthase can not be greatly influenced still, and the mutant has very high industrial value.

Modified polypeptides based on the botulinum neurotoxin A heavy chain containing the double mutation Trp-Tyr->Leu-Ser in the ganglioside binding motif Ser-X-Trp-Tyr do not bind polysialogangliosides and nerve endings. The polypeptides are useful in the preparation of nontoxic vaccines against the effects of C. botulinum infection. The modified polypeptides are also useful as vehicles for the transepithelial delivery of diagnostic and therapeutic entities, through formation of conjugates between the polypeptides and the diagnostic or therapeutic entities.

The present invention pertains to Bordetella bacteria having a double mutation, a first mutation in a gene of the Type III secretion system and a second mutation in a gene of the adenylate cyclase toxin (CyaA) locus of the bacteria so that the mutations result in no Type III secretion system, a non-functional Type III secretion system, no CyaA protein, or a non-functional CyaA protein or a combination thereof. The Bordetella bacteria double mutant is attenuated while maintaining the efficacy of the bacteria to elicit an immune response. The present invention also pertains to vaccine compositions and methods for treating and immunizing a mammal against a disease caused by infection of Bordetella bacteria or a disease caused by a pathogen.

The invention discloses an epoxide hydrolase mutant and its application. The mutant is the 108th isoleucine or the 131st aspartic acid or the 247th aspartic acid in the amino acid sequence shown in SEQ ID NO.2. Threonine is obtained by carrying out single mutation, double mutation or triple mutation; compared with the unmutated epoxide hydrolase mutant, the enzymatic activity, stereoselectivity and stability of the epoxide hydrolase mutant according to the present invention Significantly improved; Among them, the specific enzyme activity of the mutant was improved by 5.4 times compared with the original enzyme, reaching 315.2U / mg, the half-life was improved by 12.8 times, the enantioselective E value was improved by 2.1 times, and the epoxide hydrolase mutant was In the two-phase system, 800mM racemic epichlorohydrin can be catalytically resolved, and the yield of R-epichlorohydrin reaches more than 90% of the theoretical yield, and the enantioselectivity is greater than 99%.

The invention discloses an EGFR inhibitor for targetedly treating a cancer and a preparation method and application thereof.The structural formula of the EGFR inhibitor is shown in the formula I.The EGFR inhibitor can be used for preventing and / or treating the cancer such as a human skin caner or a lung cancer.The EGFR inhibitor can selectively inhibit cell lines of EGFR double mutation (EGFRT790M and L858R), and the inhibitory activity to EGFR wild type cells is weak; therefore, the EGFR inhibitor can be used for treating lung cancer patients with EGFRT790M and L858R mutation, and the side effects are fewer, wherein the side effects are caused by inhibiting wild type EGFR, for example, Afatinib.

The invention relates to a rape multiple ALS (acetolactate synthase) inhibitor type herbicide-resistant gene based on in-vitro site-specific mutagenesis and application, and belongs to the technical field of biology. The gene mALS3 is obtained by introducing a new mutant site into the ALS3 gene sequence of the herbicide-resistant rape M342, and the site is positioned at the 560th site of the ALS3 gene; the original gene is alanine with a C alkaline group at the 187th site, the C alkaline group is mutated into a T alkaline group, and valine is formed at the 187th site. The rape mALS3 gene with double mutation sites A187V and W556L is obtained through an in-vitro site-specific mutation technique. The double mutation sites are not reported in the literatures of the rape genome. After preliminary functional verification of transgenic arabidopsis, the rape mALS3 gene with double mutation sites has the advantages that the obvious resistance on multiple ALS inhibitor herbicides, such as imidazolamide, tribenuron-methyl, difluorosulfonamide and bispyribac-sodium, is realized; the resistance gene can be used for the culturing of herbicide-resistant crops.

The invention discloses a recombinant ECB (echinocandin B) deacylase mutant and an application in synthesis of ECB parent nucleus from the ECB by biocatalysis. The deacylase mutant is obtained throughsingle mutation or double mutation of the 287th and 527th sites of an amino acid sequence shown in SEQ ID No.2. AuEBDA primary structure and enzyme conformation are changed through mutation of an auebda gene sequence, and the catalytic performance of AuEBDA is improved. Influences of G287Q and R527V substitution on activity of ECB deacylase are systemically analyzed. The enzyme activity of constructed double mutant AuEBDA-G287Q / R527V is improved by 2.5 times than that of parent wild AuEBDA, and the catalytic efficiency kcat / Km is increased by 2.9 times.

The invention discloses application of a UB2 / UB3 gene in regulation and control of corn multi-spike development, and particularly relates to application of UB2 and UB3 gene double mutation in regulation and control of corn multi-spike development. According to the invention, gene editing is carried out on two targets UB2 and UB3 in corn by using a CRISPR / Cas9 transgenic technology, so that the target site of the UB2 gene has deletion of a basic group to cause frameshift mutation and deletion of large fragments of the UB3 gene. Results show that the tassel branch number of UB2 and UB3 double mutant plants is reduced, the increase of the spike number of the UB2 and UB3 double mutants is observed for the first time, new female spikes grow on spike stalks of main spikes, the multi-spike property is shown, auxiliary meristem at the lower part is also derepressed to be developed into female spikes, and phenotypes with increased aerial roots are accompanied; and it shows that after the UB2 gene and the UB3 gene are knocked out, a corn mutant plant can show a multi-spike phenotype. The invention provides a theoretical basis for genetic improvement of the corn multi-spike property has a large application prospect.

The invention, which relates to the fields of biotechnology and enzyme engineering, particularly discloses a beta-mannase mutant Man5AS11R with improved heat resistance and specific activity and an encoding gene thereof. The preparation method comprises the following steps: carrying out double mutation of K16C and D296C on beta-mannase Man5A of which the amino acid sequence is shown as SEQ ID NO.1to obtain a mutant Man5AS11R that enables the heat resistance of the mutant to be improved by 5 DEG C by being compared with Man5A; for the Man5ASamino acid sequence shown by SEQ ID NO. 2, carrying out simultaneous mutation of three points of R21V, I80D and I218E continuously to obtain a mutant Man5AS11R with the amino acid sequence shown by SEQ ID NO.4. The obtained mutant Man5AS11R has the activity that is improved by 22.5% and 31.4% respectively be being compared with the Man5A and the Man5AS on the premise that the heat resistance of the mutant Man5AS11R is kept to be unchanged. The over80% of enzyme activity of the Man5AS can be left when the Man5AS is kept for 3min at a high temperature of 80 DEG C but the Man5A can only keep 80% of enzyme activity when being kept for 3min at a temperature of 75 DEG C. Therefore, the beta-mannase mutant Man5AS11R and the encoding gene thereof have the broad development and application prospects.

The invention discloses a construction method and an application of a PHF6 <delta> and JAK3<M511I> double-mutation acute T lymphocytic leukemia mouse model. The preparation method comprises the following steps: utilizing a Vav1-Cre mouse to mate with a Phf6<fl / fl> mouse to obtain Vav1-Cre; a Phf6<fl / Y> (Phf6<- / Y>) male mouse; mating the Mx1-Cre mouse and the Phf6<fl / fl> to obtain Mx1-Cre; Phf6<fl / Y>male mouse; respectively enriching Lin-cells in bone marrow of two male mice, infecting retroviruses overexpressing GFP-JAK3M511I, and transplanting GFP + cells into receptor mice irradiated by a lethal dose through caudal vein injection, so as to construct two T-ALL leukemia mouse models. The model provided by the invention can be rapidly passed in mice, simulates the leukemia process of a T-ALL patient with PHF6 gene and JAK3 gene double mutation, and is used for targeted therapy drug screening and curative effect evaluation.

The invention provides a primer and probe combination for a digital PCR detection system for double mutations of human EGFR gene T790M and L858R, the digital PCR detection system for the double mutations of the human EGFR gene T790M and L858R, a digital PCR detection reagent kit for the double mutations of the human EGFR gene T790M and L858R, and a detection method of the double mutations of the human EGFR gene T790M and L858R. High sensitivity detection of the T790M and L858R is realized by the method of the digital PCR platform in the dual system, and so the cost is reduced and the consumption of samples is reduced, especially for ctDNA with scarce samples. Moreover, the detection of other sample types such as tumor tissues is facilitated, and certain medication guidance is provided forpatients with non-small cell lung cancer.

We disclose a cell having double mutations of the hERG gene that lead to charge reversal amino acid substitutions at residues 466 and 534 of the wild type Kv11.1 channel protein. These double charge reversal mutations result in cells having constitutively open Kv11.1 channels. Such cells could be used in a method of testing development-stage drugs and other compounds for Kv11.1 channel block activity.

The invention relates to a double mutation detection method and a reagent box for a hepatitis B virus DNA A1762T-G1764A, in particular to a double mutation detection method for detecting the A1762T-G1764A, in the DNA sequence of the hepatitis B virus by utilizing a molecular beacon probe technology and a real time PCR method as well as a reagent box for finishing the detection. The method of the invention can specially and sensitively detect the double mutation hepatitis B virus DNA of the A1762T-G1764A in a clinic blood sample.

The invention discloses a primer, probe and kit for detecting cis-trans mutation of EGFR genes C797S and T790M. The primer, probe combination and kit can accurately detect cis-double mutations of EGFRgenes C797S and T790M based on a real-time fluorescent PCR technology platform, have the advantages of fast detection speed, easiness in result interpretation, low detection reagent cost, high detection sensitivity and high specificity, and are suitable for detecting tissue sample DNAs and plasma free DNAs.

Disclosed is a new use of ibrutinib (PCI-32765). In particular, the present invention has found that ibrutinib can treat non-small cell lung cancer carrying EGFR T790M mutations and / or EGFR L858R mutations and / or EGFR delE746_A750 mutations, especially non-small cell lung cancer carrying EGFR L858R and EGFR T790M double mutations.

The invention relates to a double mutation detection method and a reagent box for a hepatitis B virus DNA A1762T-G1764A, in particular to a double mutation detection method for detecting the A1762T-G1764A, in the DNA sequence of the hepatitis B virus by utilizing a molecular beacon probe technology and a real time PCR method as well as a reagent box for finishing the detection. The method of the invention can specially and sensitively detect the double mutation hepatitis B virus DNA of the A1762T-G1764A in a clinic blood sample.

The invention provides a method for obtaining a cucumber germplasm material with high powdery mildew resistance through multi-gene editing. CsMLO1, CsMLO8 and CsMLO11 genes are enabled to produce double mutation or three mutation, or amino acids coded by the CsMLO1 gene, the CsMLO8 gene and the CsMLO11 gene are enabled to produce double mutation or three mutation together, so that the powdery mildew resistance of cucumbers is improved. Compared with the prior art, the CsMLO1 gene, the CsMLO8 gene and the CsMLO11 gene are adopted to jointly regulate and control the powdery mildew resistance of cucumbers, and a powdery mildew complete resistance material can be obtained; and compared with the prior art, the method has the advantages that common mutation of the CsMLO1 gene, the CsMLO8 gene and the CsMLO11 gene is realized by adopting a CRISPR / Cas9 multi-gene editing technology, the obtained germplasm material is a transgenic trace-free mutant material without an exogenous transgenic fragment, and the transgenic safety risk is lower.

We disclose a cell having double mutations of the HERG gene that lead to charge reversal amino acid substitutions at residues 466 and 534 of the wild type Kv11.1 channel protein. These double charge reversal mutations result in cells having constitutively open Kv11.1 channels. Such cells could be used in a method of testing development-stage drugs and other compounds for Kv11.1 channel block activity.

Compositions and methods are provided for diagnosis, prognosis and treatment of AR-related diseases, such as prostate diseases, such as prostate cancer, breast cancer, and many other diseases. In particular, a novel and clinically relevant mutation at position 876 of the androgen receptor (AR) has been identified. Drug sensitivity can be predicted and therapeutic regimens can be planned on the basis of the presence or absence of this mutation. Polypeptides comprising, antibodies to, and polynucleotides encoding the mutant AR can be used to identity novel treatments. A double mutation in AR at positions 741 and 877 is also shown to be useful for patient stratification.

The invention discloses a nitrilase mutant and application thereof in synthesis of benzoylformic acid compounds. The mutant is obtained by performing single mutation or double mutation on tryptophan at the 144th site or tyrosine at the 191st site of an amino acid sequence as shown in SEQ ID NO.2. The invention further discloses a preparation method of the nitrilase mutant. Compared with non-mutated wild nitrilase, the nitrilase mutant disclosed by the invention has the advantages that the enzyme activity (1.2 U / L) is obviously improved and reaches 162.5 U / L; and when benzoyl nitrile is catalyzed to be hydrolyzed to produce benzoylformic acid, the highest raw material conversion rate can reach 96%, and the method has a very great industrial application prospect.

The present invention provides a mutant of Perakine reductase and its application. The mutant is obtained by the following single mutation or combined double mutation of 126 and 127 in the amino acid sequence of wild-type Perakine reductase at position 126 or 127: H126G / R / I / C / P / K / N / W / A / Y, R127M / P / N / S / F, where " / " stands for "or". Based on the three-dimensional crystal structure and sequence comparison, the present invention adopts the point-saturation mutation technology to carry out molecular transformation on Perakine reductase, and screens to obtain the mutant capable of selectively reducing the enone double bond. The method of the present invention as a catalyst for selectively reducing enone double bonds has the advantages of simple reaction steps, mild reaction conditions, environmental friendliness, high catalytic efficiency, simple enzyme purification, good stability, and strong stereoselectivity. Bond reduction and the synthesis of related saturated ketone drugs have good application prospects.

The invention discloses a linoleate isomerase mutant and application thereof, and the linoleate isomerase mutant is obtained by mutation of 62th-site methionine of linoleate isomerase with an amino acid sequence as shown in SEQIDNO:1 into alanine or glycine, and mutation of 295th-site serine into alanine, leucine or valine. The enzyme activity for catalysis of linoleic acid for preparation of trans-10,cis-12 conjugated linoleic acid of the linoleate isomerase mutant can be greatly improved compared with that of a wild-type enzyme, especially during double mutation such as M62A / S295L, M62A / S295A, M62A / S295V, M62G / S295L, M62G / S295A and M62G / S295V, the enzyme activity can be increased by about 10 times, and the linoleate isomerase mutant has good prospects for industrial application.