93results about How to "Efficient and stable expression" patented technology

The invention provides an insect-resistant gene Cry1F-t and application thereof. A Cry1F gene is completely modified to obtain the Cry1F-t insect-resistant gene as shown by SEQ ID NO: 1; an AdhI enhancer is added in the front of the Cry1F-t insect-resistant gene; the Cry1F-t insect-resistant gene is put on an expression vector including a strong promoter 35S; and the Cry1F-t gene is transferred into a corn genome through an agrobacterium-mediated genetic transformation method to obtain a transgenic material including the Cry1F-t. Proved by an experiment, the Bt toxalbumin modified and synthesized by the invention has an obvious insect killing effect on corn borers; and the Cry1F-t and a target protein expressed by the Cry1F-t can be stably and efficiently expressed in a corn, so that the Cry1F-t and the target protein are used for culturing a Cry1F-t transgenic insect-resistant corn. The Bt insect-resistant gene Cry1F-t can be also applied in the improvement of the insect resistance of other farm crops, fruit trees or vegetables and the like, and an effective approach is provided for solving insect pests in the production of plants such as the corn.

The invention relates to a novel EPSPS (5-enolpyruvyl-shikimate-3-phosphate synthase) gene, an expression product of the EPSPS gene, a herbicide-resistant gene CTP2-EPSPS (chloroplast transit peptide 2-EPSPS), an expression product and an expression vector of the herbicide-resistant gene CTP2-EPSPS and application of the expression product and the expression vector of the herbicide-resistant gene CTP2-EPSPS. Nucleotide sequences of the EPSPS gene are shown as SEQ ID No.2, amino acid sequences of encoding proteins of the expression product of the EPSPS gene are shown as SEQ ID No.3, the herbicide-resistant gene CTP2-EPSPS contains chloroplast transit peptide CTP2 and the EPSPS gene, nucleotide sequences of the herbicide-resistant gene CTP2-EPSPS are shown as SEQ ID No.5, and the expression product and the expression vector of the herbicide-resistant gene CTP2-EPSPS can be applied to cultivating glyphosate-resistant transgenic corn. The EPSPS gene, the expression product of the EPSPS gene, the herbicide-resistant gene CTP2-EPSPS, the expression product and the application vector of the herbicide-resistant gene CTP2-EPSPS and the application have the advantages that the herbicide-resistant gene can be stably expressed in crop and is high in expression amount, good herbicide-resistant effects can be realized, the corn or other plants can be transited, accordingly, herbicide-resistant characteristics of plants such as the corn can be improved, and an effective way can be provided for improving the herbicide-resistant characteristics of the plants such as the corn during production.

The invention relates to an encephalitis b virus non-structural protein NS1 truncated mutant as well as a coding gene and application thereof, which belongs to the technical field of viruses. According to the encephalitis b virus non-structural protein NS1 truncated mutant, on the basis of original NS1, two strong hydrophobic zones on an end C are deleted, a truncated mutant NS1 delta63 gene with63 amino acids on the end C being deleted, then the truncated mutant NS1 delta63 gene is cloned to a prokaryotic expression vector pET-28a(+), a recombinant expression vector pET28a-NS1delta63 is obtained, escherichia coli is converted, and by virtue of the induction of IPTG, the NS1 delta63 can be efficiently and stably expressed in the escherichia coli. The NS1 delta63 protein expressed in the Escherichia coli purifies and immunizes a mouse, and the NS1 anti-serum titer of the immunized mouse reaches 1:12150 by virtue of ELISA detection. The protection capability test of the immunized mouseproves that the NS1 delta63 has the protection activity. The NS1 delta63 can lay a foundation for later research of the virus vaccine and diagnosis kit.

The invention provides an insecticidal protein Cryl A. 301 which has 1) an amino acid sequence shown by SEQ ID No.2; or 2) an amino acid sequence which is obtained by substituting the amino acid sequence shown by SEQ ID No.2 or decreasing and or increasing one or more amino acid(s) of the amino acid sequence shown by SEQ ID No.2, and has the same function. The experiment in vitro proves that the reformed and synthesized Bt gene toxin production protein has remarkable insecticidal effect for european corn borer. The insecticidal protein Cryl A. 301 can be efficiently expressed in monocotyledons, thus being used for producing insect-resistant transgenic plants.

The invention belongs to the field of biotechnology and botany, and particularly provides a construction method of a rolling-circle replication recombinant vector for heterologous protein expression,the recombinant vector and an application of the recombinant vector. According to the construction method provided by the invention, the high-efficiency rolling-circle replication transient expressionvector mainly based on the sequence of a sweet potato leaf curl virus (SPLCV) is constructed, so that duration time and expression abundance of heterologous nucleic acids and proteins on plants are remarkably improved, and the results of the vector have high repeatability and reliability. The recombinant vector provided by the invention has a molecular tool vector which has an extremely long expression time to heterologous proteins (longer than 20 day compared with the expression time of a common transient expression T vector) and has an extremely high expression abundance (larger than 30-fold expression compared with a common transient expression T vector), and the recombinant vector endows different plants (tobaccos, sweet potatoes and morning glory) with heterologous protein expressioncapability, and makes up the blank of related plants for utilizing transient expression to study gene functions.

The invention provides a liver cancer tissue specific RNA (Ribose Nucleic Acid) interference system, as well as a construction method and an application method thereof. The liver cancer tissue specific RNA interference system comprises an AFP (Alpha Fetal Protein)-Cre vector and an LoxP-shRNA vector which are both constructed by lentiviral vectors, wherein the AFP-Cre vector contains an AFP promoter and a Cre recombinase gene placed at the downstream of the AFP promoter; the LoxP-shRNA vector contains a reconstructive H6 promoter and an shRNA sequence placed at the downstream of the U6 promoter; the reconstructive U6 promoter contained in the LoxP-shRNA vector is the U6 promoter in which a structure, capable of indicating whether the AFP-Cre / LoxP-shRNA system is in work, is inserted inside; and the structure, capable for indicating whether the AFP-Cre / LoxP-shRNA system is in work, is shown as LoxP-CMV-eGFP-LoxP. The RNA interference system, provided by the invention, has the characteristics of being high in liver cancer tissue specificity, long-lasting in effect, high in efficiency, excellent in indication performance, and simple and convenient in operation; and the liver cancer tissue specific RNA interference system enables the liver cancer RNA interferential targeted treatment of liver cancer, provides high convenience to relative research and analysis, and can be widely applied to the treatment of the liver cancer and the basic research.

The invention discloses a method for constructing eukaryon Hansenula polymorpha with recombinant hepatitis B virus genes and a method for producing hepatitis B surface antigens, and belongs to the technical field of bioengineering. The method for constructing the eukaryon Hansenula polymorpha includes steps of 1, constructing plasmids pHPZF1.0-ZS with the recombinant hepatitis B virus HBsAg genes; 2, screening the plasmids to obtain the eukaryon Hansenula polymorpha engineering bacteria with the recombinant hepatitis virus HBsAg genes and the highest expression level. The methods have the advantages that HBsAg recombinant proteins can be stably and efficiently expressed in a methanol induction mode by Hansenula polymorpha engineering bacterial strains of the hepatitis B surface antigens which can be obtained by the aid of the methods, and the methods are suitable for producing the HBsAg recombinant proteins on a large scale.

The invention discloses a high-throughput expression method of alkaline bacterial laccase in yeast. The high-throughput expression method comprises the following steps: (A) a pichia pastoris expression vector carrying with alkaline bacterial laccase genes is constructed; (B) positive transformants are screened; (C) the yeast expression vector is linearized; (D) the yeast galvanic competence is prepared; (E) recombinants are expressed and identified; (F) fusion proteins are purified; (G) enzyme kinetic parameters are determined. The high-throughput expression method helps modelers to finish the compositional modeling of models more efficiently and reasonably through the model discovery based on the semantics, so that the problems of easy formation of inclusion bodies by the alkaline bacterial laccase in the prokaryotic expression system, low yield and low enzymatic activity are solved.