52 results about "Prokaryotic expression" patented technology

This invention discloses a methanogenic lyase homologous to PeiR lyase and its applications. The PeiR lyase protein, targeting the peptide bonds of the methanogenic cell wall, participates in the biological process of hydrolyzing the archaea cell wall, effectively killing methanogens and reducing methane production. Proteins homologous to this lyase possess potential methane-reducing potential. This invention utilizes a series of bioinformatics software to identify protein sequences encoded by methanogenic viruses from a large amount of rumen microbial virome sequencing data, and analyzes the structural homology between these proteins and PeiR. Ultimately, this invention successfully obtained a series of methanogenic lyases homologous to PeiR. To achieve the expression of these lyases, this invention designed multiple primers to synthesize the target sequences and successfully expressed them in a prokaryotic expression system. In vitro gas production experiments with the crude enzyme solution showed that these lyases are effective in reducing methane production.

This invention discloses a chikungunya virus genome fragment armored RNA reference, its preparation method, and its application. The preparation process involves the following steps: (1) constructing the prokaryotic expression backbone vector pACYCAR; (2) cloning the CKV genome fragment CKA (nt17-2276); (3) constructing the prokaryotic expression vector pACYCAR-CKA; (4) inducing the expression of armored RNA; and (5) purifying the armored RNA. This invention solves the technical problems of high biosafety risk, poor stability, and insufficient gene fragment coverage in existing CKV nucleic acid detection references. It fills the gap in the lack of high-quality references for CKV detection.

The application discloses a novel lactoferrin inducible soluble expression system and a construction method thereof, and belongs to the technical field of biotechnology and genetic engineering, and the specific scheme is that human lactoferrin (LF) is expressed by probiotic Escherichia coli Nissle1917, the protein is purified after the expression position is determined, and anti-inflammatory activity is determined. The LF expression system constructed by the application provides a construction method of a prokaryotic expression stable human lactoferrin expression vector, the vector is introduced into probiotic Escherichia coli Nissle1917, soluble target recombinant human lactoferrin is obtained after induction fermentation expression, two-step purification and identification, and the application has the characteristics of high safety, low cost, easy operation and the like.

This invention discloses a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) antibodies and its applications, belonging to the field of animal disease detection technology. The kit uses a mutant nucleocapsid protein (N protein) of the PRRSV-2 class NADC30 strain CHsx1401 as the coating antigen; the N protein mutant has mutations at amino acid positions 23, 75, and / or 90 compared to the wild-type N protein. High purity is achieved through optimized prokaryotic expression systems and purification processes. Under optimal detection conditions, the kit exhibits strong specificity, high sensitivity, and good reproducibility, with significantly superior detection performance compared to existing commercial kits. It can be used for PRRSV-2 antibody detection, epidemiological surveys, and disease control.

The application discloses a CMV virus-like particle for producing a VLP recombinant vaccine and a preparation method thereof, the CMV virus-like particle is obtained by cloning a C-terminal G4SLPETG modified plant virus cucumber mosaic virus CMV capsid gene into a prokaryotic expression vector to obtain a recombinant expression vector, the recombinant expression vector is transfected into E. coli BL21 (DE3), and expression is obtained through the recombinant E. coli BL21 (DE3); and the amino acid sequence of the C-terminal G4SLPETG modified CMV is SEQ ID NO. 1. Test proves that the recombinant strain constructed by the application is stable to expression of an exogenous protein. The recombinant protein expressed by the application is used for preparing a virus-like particle for antigen coupling, the coupling efficiency is high, the antigen purity is high, the safety is good, the virus-like particle is not pathogenic to mice and other animals, and the safety evaluation is easy.

This invention relates to the field of genetic engineering technology, specifically disclosing a polyclonal antibody against citrus HIPP7 protein, its preparation method, and its applications. The method includes selecting amino acid 1-246 of the HIPP7 protein sequence for prokaryotic expression, synthesizing the sequence and ligating it into the vector pET41a to construct pET41a-HIPP7, transforming it into *E. coli*, culturing it in LB liquid medium containing kanamycin, inducing expression with IPTG, isolating and purifying the protein, and preparing a polyclonal antibody against citrus HIPP7 protein by immunizing rabbits with the HIPP7 protein. The polyclonal antibody against citrus HIPP7 protein obtained by this invention can specifically detect citrus HIPP7 protein and can be used to detect the accumulation of HIPP7 protein in different citrus tissues and under different treatment conditions, as well as to detect the accumulation of HIPP7 protein in transgenic plants.

This invention discloses a cPGES protein based on immunological screening of a bovine cysticercosis cDNA prokaryotic expression library and its applications, belonging to the field of biochemistry. The purpose of this invention is to provide a method for the detection, treatment, and prevention of bovine cysticercosis. This invention provides a bovine cysticercosis antigen, the amino acid sequence of which is shown in SEQ ID NO.4. This provides a new candidate antigen for the development of novel vaccines and specific diagnostic reagents.

This invention discloses a prokaryotic expression system and preparation method for a linear precursor peptide of zinconotide. This invention belongs to the field of genetic engineering, specifically relating to a prokaryotic expression system and preparation method for a linear precursor peptide of zinconotide. The recombinant vector of this invention contains a zinconotide-encoding gene expression cassette, which includes the coding sequences of the zinconotide-encoding gene and a fusion tag. The nucleotide sequence of the zinconotide-encoding gene is positions 1447-1521 of Sequence 1, and the nucleotide sequence of the fusion tag is positions 256-1428 of Sequence 1. This invention achieves high-volume soluble expression in an *E. coli* expression system to obtain a recombinant linear precursor peptide of zinconotide. The correct target recombinant linear peptide is obtained by collecting bacterial cells and using a two-step purification process, resulting in high yield, easy preparation, and environmentally friendly production.

This invention relates to the field of recombinant protein technology, specifically to an α-hemolysin recombinant protein and its prokaryotic expression method. A method for preparing a high-purity, tag-free α-hemolysin monomer is as follows: an expression vector encoding the fusion protein of SEQ ID NO.7 is constructed and transformed into competent cells to obtain engineered bacteria; after culturing and inducing expression, the bacterial cells are lysed and centrifuged to obtain the supernatant; the fusion protein eluate is obtained by a first affinity chromatography on a nickel column, dialyzed, and digested with HisSUMO protease; a second nickel column chromatography is performed, and the flow-through is collected to obtain a high-purity, tag-free α-hemolysin monomer. This technical solution can solve the technical problems of existing prokaryotic expression of α-hemolysin recombinant proteins, such as severe inclusion body formation, complex purification processes, insufficient product activity, and large batch-to-batch variability, thereby obtaining a high-purity, highly active, and structurally intact α-hemolysin monomer, which has ideal application value.

The present application belongs to the technical field of bioengineering. The present application relates to a recombinant protein, which comprises two dominant antigen epitopes of feline calicivirus (FCV) protein. In order to improve the yield of the recombinant protein in a prokaryotic expression system, the amino acid sequence of the recombinant protein is converted into a corresponding nucleotide sequence by using E. coli preferred codons, the nucleotide sequence is chemically synthesized, and a recombinant expression vector is constructed. The present application also relates to the establishment of a phage library by immunizing mice with the recombinant protein, the screening of a single-chain antibody (scfv) sequence corresponding to the feline calicivirus recombinant protein by panning, the construction of a complete mouse IgG1 antibody sequence expression vector from the obtained scfv sequence, the expression of a monoclonal antibody by instantaneously transferring HEK293F cells, the purification of the monoclonal antibody, and the labeling of europium ions (Eu 3+ ) in the monoclonal antibody, respectively. The optimal monoclonal antibody pairing combination is determined by orthogonal experiments, and the monoclonal antibody can be used for the early diagnosis of upper respiratory tract infection in cats.

The application belongs to the field of animal bacteriology and molecular biology technology, and relates to preparation of a hybridoma cell and application thereof. A monoclonal antibody is prepared through intracellular Lawsonia intracellularis outer membrane protein OmpA expressed by prokaryotes, and IFA, WB and blocking ELISA and other immunological detection methods for detecting or diagnosing Lawsonia intracellularis are established through the expressed intracellular Lawsonia intracellularis outer membrane protein OmpA and the prepared monoclonal antibody. The blocking ELISA for detecting serum antibodies of Lawsonia intracellularis can detect serum samples of suspected Lawsonia intracellularis infection from different species, and does not exist cross reaction with positive serum of common pig pathogens, has high sensitivity and specificity, is good in repeatability, and is suitable for high-throughput detection of serum samples.

This invention relates to an anti-phospholipase A2 receptor polyclonal antibody, its preparation method, and its application. The preparation method includes immunizing animals with phospholipase A2 receptor protein as an antigen and collecting serum; separating and purifying the serum to obtain the anti-phospholipase A2 receptor polyclonal antibody; the amino acid sequence of the phospholipase A2 receptor protein includes the sequence shown in SEQ ID NO.1. This invention designs specific antigens for the preparation of polyclonal antibodies, enabling efficient preparation of polyclonal antibodies that specifically recognize the phospholipase A2 receptor. It has also been found that polyclonal antibodies prepared using eukaryotically expressed antigens, compared to those prepared using prokaryotically expressed or peptide-derived antigens, can more stably and efficiently construct an anti-PLA2R-associated membranous nephropathy (PLA2R MN) animal model.

A bacteriophage lytic enzyme and its application. The name of the bacteriophage lytic enzyme is PlyNJ3; the gene fragment encoding the lytic enzyme PlyNJ3 is amplified from streptococcus suis by PCR, and a prokaryotic expression vector plasmid pET32a-PlyNJ3 containing the PlyNJ3 gene is constructed, the recombinant plasmid is obtained by transforming DH5 alpha competent cells, and the protein is massively expressed in an escherichia coli expression system. Then, the lytic enzyme PlyNJ3 is purified by affinity purification medium, the high-efficiency expression of the lytic enzyme PlyNJ3 in vitro is realized, and the lytic effect of the lytic enzyme PlyNJ3 on different serotypes of streptococcus suis, streptococcus agalactiae and other common streptococci is verified in vitro. The bacteriophage lytic enzyme PlyNJ3 prepared by the application can selectively lyse target pathogenic bacteria, and has a good application prospect in the development of new bacteriophage drugs.

The application discloses an anti-goat CD207 single-chain antibody and a preparation method and application thereof. Based on nucleotide sequences of heavy chain and light chain variable regions of obtained anti-goat CD207 monoclonal antibodies, a single-chain antibody capable of specifically combining with goat CD207 protein is obtained through single-chain antibody gene construction and prokaryotic expression, a heavy chain variable region amino acid sequence of the single-chain antibody is shown as SEQ.ID.NO.1, and a light chain variable region amino acid sequence is shown as SEQ.ID.NO.2. The anti-goat CD207 single-chain antibody of the application has good reactivity with goat CD207 protein.

The application discloses a method for preparing a reproductive vaccine based on a novel carrier protein coupled GnRH polypeptide and a product, wherein the amino acid sequence of the carrier protein is shown as SEQ ID NO.1, the gene sequence for coding the carrier protein is shown as SEQ ID NO.2, the gene for coding the carrier protein is subjected to prokaryotic expression, and then subjected to affinity chromatography, anion exchange chromatography purification, TEV enzyme cutting to remove a tag protein, and then subjected to affinity chromatography and molecular sieve to obtain the purified carrier protein, and finally covalently coupled with a GnRH polypeptide to obtain the reproductive vaccine, wherein the reproductive vaccine prepared by the application has a contraceptive effect, and high-quality, low-cost recombinant protein vaccines can be obtained on a large scale for commercialization.

The application provides a soluble expression method, an expression vector and an expression engineering bacterium of Japanese medical leech destabilizing enzyme, and belongs to the field of prokaryotic expression technology. The application provides a novel soluble prokaryotic expression method of destabilizing enzyme. Through accurate design of vector construction elements and selection of expression hosts, the soluble efficient expression of the destabilizing enzyme in supernatant is realized, the cumbersome steps of inclusion body renaturation and the risk of folding errors are avoided, the natural conformation and biological activity of the target enzyme are ensured, the subsequent purification process is greatly simplified, the preparation efficiency is improved, a feasible technical scheme is provided for large-scale preparation and practical application of the destabilizing enzyme, and the application has important industrial application value.

This invention relates to the field of biomedical technology, specifically disclosing a three-component recombinant subunit vaccine composition for Mycoplasma pneumoniae and its application. The vaccine composition comprises key antigenic proteins of Mycoplasma pneumoniae (MP): P1 protein, P40 / 90 protein, and an E132A attenuated mutant of community-acquired respiratory distress syndrome (CARDS) toxin (CARDS-Mut), optionally combined with Al(OH)3 single adjuvant or Al(OH)3+CPG double adjuvant. The recombinant protein was successfully purified using a prokaryotic expression system, and validated in a mouse model. The vaccine induces high-titer specific IgG antibodies, significantly alleviates lung tissue pathological damage, reduces bacterial load, and inhibits inflammatory factor secretion after challenge, with good safety. This invention solves the problem of high toxicity of CARDS toxin, providing an efficient and safe vaccine strategy for the prevention and control of MP infection.

The application belongs to the technical field of genetic engineering antibodies, and particularly relates to an antibody for non-competitive detection of aflatoxin B1 and application thereof. The antibody comprises a nanobody of an aflatoxin B1 antigen-antibody immune complex and a monoclonal antibody against aflatoxin B1. The amino acid sequence of the nanobody of the aflatoxin B1 antigen-antibody immune complex is shown in SEQ ID NO. 1. The monoclonal antibody against aflatoxin B1 comprises a heavy chain and a light chain. The amino acid of the heavy chain is shown in SEQ ID NO. 10, and the amino acid of the light chain is shown in SEQ ID NO. 11. The antibody for non-competitive detection of aflatoxin B1 has low cost, can be prepared in a large amount through a prokaryotic expression system, is simple to prepare, has strong operability, and can be applied to non-competitive immunological analysis of aflatoxin B1.

The application belongs to the technical field of biology, and particularly relates to a grass carp reovirus (GCRV-Ⅱ type) subunit recombinant vaccine, a preparation method and application. 274‑367 , VP5 105‑336 , NS38 143‑338 sequence fragments, and the three sequence fragments are recombined and expressed in a prokaryotic expression vector to prepare the subunit recombinant vaccine. The recombinant vaccine has high safety, strong activity and broad spectrum, and has good application potential.

This invention discloses the chitin synthase 1 (LmCHS1) gene of the leafminer moth, its dsRNA, its synthesis method, and its insect-resistant applications. Through transcriptome analysis, specific primer pairs for the open reading frame of the LmCHS1 gene were designed and synthesized. The LmCHS1 gene sequence was obtained through gene cloning (nucleotide sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2). The gene fragment with sequence SEQ ID NO: 3 was selected as the RNAi target sequence. Primer pairs SEQ ID NO: 6 and SEQ ID NO: 7 with restriction enzyme sites were designed and synthesized. PCR amplification was performed, and a prokaryotic expression vector was constructed to induce dsRNA expression. Feeding leafminer moth larvae with dsRNA interfered with LmCHS1 gene expression. After 24 hours, abnormalities were observed in the larvae's body surface, with some larvae failing to complete molting or dying due to an inability to form a normal body surface after molting. After 96 hours, the larval mortality rate was 72.22%, compared to 40% in the GFP control group, demonstrating a significant effect. This invention has broad application prospects in the control of the leaf borer pest in forestry due to its high efficiency, specificity, and safety.

The application discloses a preparation method and application of a recombinant collagenase type I and a mutant thereof. A mutant of the collagenase type I is provided in the application, and the mutant has good alkali resistance and can be applied to enzymatic extraction of fish skin waste. The application further provides an expression method of the recombinant collagenase type I and the mutant thereof based on a prokaryotic expression system. The expression method can improve the expression amount of soluble protein, and the product has high activity and is suitable for industrial amplification production.

The application discloses a method for expressing BirA enzyme by using prokaryotic Escherichia coli and purifying the BirA enzyme, and relates to the technical fields of genetic engineering and protein engineering. A recombinant expression vector for expressing the BirA enzyme is a pET-28A-BirA plasmid which is constructed by inserting a BirA enzyme coding gene into a multiple cloning site of a pET-28A vector. An 8× histidine tag coding nucleotide sequence is fused to a C terminal of the BirA enzyme coding gene. The nucleotide sequence of the BirA enzyme coding gene is shown as SEQ ID NO:1. The prokaryotic expression system is simple in culture condition, short in cycle, and low in cost, and is suitable for large-scale preparation. The 8× histidine tag is introduced into the C terminal of the BirA enzyme gene, so that the expression product can be purified by one-step nickel column affinity chromatography, and the purification process is simple and high in efficiency.

This invention discloses a bivalent subunit vaccine against Candida albicans, its preparation method, and its application, relating to the field of biomedical technology. The bivalent subunit vaccine comprises recombinant Als3 protein and recombinant Cht3 protein as immunogens. The recombinant Als3 protein is obtained based on the sequence shown in SEQ ID NO:1, through site-directed mutagenesis of the N-glycosylation site and weak epitopes LDGTSASPG and IVIVATTRT. The recombinant Cht3 protein is obtained based on the sequence shown in SEQ ID NO:2, through site-directed mutagenesis of the weak epitopes TTSVTSSET and YTANGILQC to enhance immunogenicity. This invention overcomes the bottleneck of prokaryotic expression of Cht3 antigen through a combination strategy of antigen extraction, expression strain optimization, mild induction, and multiple site-directed mutagenesis, obtaining a high-purity, highly immunogenic antigen. Furthermore, the bivalent combination of Als3 and Cht3 produces a synergistic immune effect, significantly improving the protective effect against Candida albicans infection. Simultaneously, the process is simple and easy for industrial production.

The application relates to the field of sweeteners, in particular to a method for expressing and purifying truffle sweet protein HTS in Escherichia coli. A recombinant expression vector coding truffle sweet protein HTS is introduced into a competent cell of Escherichia coli for protein induction expression; the recombinant expression vector coding truffle sweet protein HTS comprises a basic vector and a truffle sweet protein HTS coding gene inserted into the basic vector; the amino acid sequence of the truffle sweet protein HTS is shown in SEQ ID NO: 3. The application limits the conditions such as the concentration of an inducer, induction time and induction temperature, centrifugal collection of bacterial liquid after induction expression, ultrasonic crushing treatment and collection of supernatant, and through affinity chromatography and gel chromatography, the prokaryotic expression and purification of truffle sweet protein HTS can be realized, and a large amount of truffle sweet protein HTS with a purity of more than 95% can be obtained within two days, and the truffle sweet protein HTS is easy to mass-produce.

This invention belongs to the field of biomedicine, specifically relating to a method for the expression and purification of recombinant human tropoelastin. More specifically, it relates to a method for efficiently expressing human tropoelastin using *E. coli* as a host by constructing an expression plasmid containing a T7 promoter, and obtaining a high-purity product through a series of purification processes. The technical problem to be solved by this invention is to provide a human tropoelastin with intact structure and high biological activity that can be efficiently expressed in a prokaryotic expression system, as well as its preparation method and applications.

The application discloses a kind of targeting cat calicivirus VP1-CDE protein chicken source single-chain antibody and its preparation and application, belong to biotechnology field.The single-chain antibody is selected from X2B2, X2E1 Or X2G5, its heavy chain variable region and light chain variable region amino acid sequence are respectively as shown in SEQ ID NO:1-6.The application is prokaryotic expression and purified VP1-CDE protein as antigen immunization chicken, amplifies VH And VL Gene and is spliced into scFv gene by (Gly4Ser) 3 linker, constructs library capacity not less than 5.25×10 7 CFU / mL phage display library, specific single-chain antibody clone is obtained by solid-phase panning, and its binding activity is verified by Western blot, ELISA, indirect immunofluorescence test after eukaryotic expression and purification.The results show that the three single-chain antibodies can specifically bind VP1-CDE protein and FCV virus particles, and there is no cross reaction, which provides key experimental materials for basic research of cat calicivirus, can be used for developing FCV rapid diagnostic reagent or kit, also provides candidate molecules for the development of FCV infection treatment drugs, and has important theoretical significance and application value.

The application belongs to the technical field of biological detection, and discloses a capripoxvirus specific competitive ELISA detection kit and application thereof. The application discloses a hybridoma cell strain GTPV-N1P-4H6, and realizes high-efficiency soluble expression of GTPV-N1 based on a prokaryotic expression system. After immunizing mice with GTPV-N1 as an immunogen, a GTPV positive hybridoma cell strain GTPV-N1P-4H6 capable of stably secreting antibodies is successfully prepared and screened after cell fusion. The ELISA titer of the antibody obtained after the monoclonal antibody secreted by the cell strain is purified is 1:64000, the heavy chain of the antibody is IgGa2 subtype, and the light chain is Kappa chain. Based on the antibody, high-efficiency detection of capripoxvirus N1 protein can be realized.