Gene sequence of recombinant expression vector and construction method thereof

Abstract

The invention provides a gene order of a recombinant expression carrier and a construction method thereof, belonging to the technical field of biological engineering. In the method, with the PCR method applied, pyrobest DNA polymerase, upstream primer and downstream prime are adopted to amplify nEbpS protein target gene from a staphylococcus aureus reference culture genome; the nEbpS protein target gene is cloned to a cloning carrier, thus obtaining a recombinant cloning carrier; a competent host cell is first transformed by the recombinant cloning carrier , then filtration and appraisal are carried out on the competent host cell to obtain recombinant plasmid; the recombinant plasmid is integrated into an expression carrier to obtain a recombinant expression carrier, then the recombinant expression carrier is transformed into colon bacillus M15 to obtain EbpS protein under the induction of IPTG; affinity chromatography is adopted to purify the EbpS protein to prepare emulsified antigen; the emulsified antigen is adopted to immunize host, host blood serum is collected and EbpS antibody preparation is prepared. The gene order obtained by adopting the high-fidelity enzyme of the invention witnesses no mutation, obtains high-precision gene fragments and ensures accuracy.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a gene sequence of a recombinant expression vector and a construction method thereof. Background technique [0002] Staphylococcus aureus is a common Gram-positive bacterium with strong pathogenicity. It is the main pathogen of secondary infection and sepsis, gastrointestinal infection, food poisoning outbreak and other zoonotic diseases. Staphylococcus aureus is the most common pathogenic bacteria in human suppurative infection, which can cause local suppurative infection, pneumonia, pseudomembranous enteritis, pericarditis, etc., and even systemic infection such as sepsis and sepsis. The pathogenicity of Staphylococcus aureus mainly depends on the toxins and invasive enzymes it produces. [0003] Studies have found that Staphylococcus aureus mainly binds to the extracellular matrix of the host through the elastin fibronectin (EbpS) on its surface, thereby causing infect...

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Problems solved by technology

[0004] Under the existing technical conditions, when using PCR technology to amplify the expression gene of the elastic fiber binding protein (EbpS), common Taq enzyme is generally used, and then the gene sequence obtained by PCR is sequenced and compared with the gene sequence in the gene bank. It shows that the amplified target gene produces a mutated sequence, and the mutation rate of this process is often relatively high, which makes it difficult to obtain the target gene and invisibly increases the economic input of experiments and materials

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