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Fusion protein TgMEP as well as preparation method and application thereof

A fusion protein and sequence technology, applied in the field of genetic engineering, can solve the problems of high preparation cost, difficult standardization, and low diagnostic efficiency of toxoplasmosis diagnostic antigen, and achieve the effect of simple preparation, good sensitivity and specificity

Inactive Publication Date: 2012-12-19
THE SECOND AFFILIATED HOSPITAL OF NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to solve the shortcomings of current toxoplasmosis diagnostic antigens, such as low diagnostic efficiency, high preparation cost and difficulty in standardization, the present invention uses genetic engineering technology to construct and induce the expression of recombinant toxoplasma multi-epitope fusion protein TgMEP, which solves the shortcomings of the prior art

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  • Fusion protein TgMEP as well as preparation method and application thereof
  • Fusion protein TgMEP as well as preparation method and application thereof
  • Fusion protein TgMEP as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1 Expression, purification and immunoreactivity detection of Toxoplasma gondii multi-epitope peptide recombinant fusion protein TgMEP

[0021] [1] Screening and identification of B cell epitopes of the main diagnostic antigen of Toxoplasma gondii

[0022] Using molecular biology software BioSun, DNAstar combined with Hopp&woods hydrophilicity parameters, accessibility parameters, polarity parameters, flexibility parameters and secondary structure parameters to analyze the major surface antigen 1 (SAG1) and major The B cell epitopes of surface antigen 2 (SAG2), major surface antigen 3 (SAG3), tachyzoite surface antigen P35, compact granule protein 1 (GRA1), and compact granule protein 6 (GRA6) were analyzed; each antigen molecule was selected Two predicted B cell epitopes, two complementary oligonucleotide single strands were designed according to the sequence of each epitope, annealed to form double strands and then cloned into the prokaryotic expression vector ...

Embodiment 2

[0032] Example 2 Application of recombinant fusion protein TgMEP in toxoplasmosis diagnostic kit

[0033] [1] Establishment of recombinant fusion protein TgMEP ELISA

[0034] Orthogonal experiments were used to determine the best detection conditions for the enzyme-linked immunosorbent assay for the detection of toxoplasmosis IgG and IgM antibodies, and the coating concentrations of the recombinant fusion protein TgMEP for the detection of IgG and IgM antibodies were respectively: 1 μg / ml and 2 μg / ml. The diagnostic cut-off value was the mean of 15 negative sera plus 2 standard deviations, and the diagnostic cut-off values ​​of ELISA for IgG and IgM antibody detection were 0.14 and 0.16, respectively. The specific detection steps are as follows: Coat polystyrene enzyme-labeled strips with 100 μl / well of carbonic acid buffer containing recombinant fusion protein, overnight at 4°C, block with PBST containing 5% skimmed milk powder at 37°C for 1 hour, and wash the plate three ti...

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Abstract

The invention relates to a fusion protein TgMEP as well as a preparation method and application of the fusion protein TgMEP. An amino acid sequence of the fusion protein TgMEP is shown in a SEQ ID NO.2. The construction and preparation method comprises the following steps of: optimizing a gene sequence of the TgMEP to obtain a sequence shown in a SEQ ID NO.1; synthetizing the optimized gene sequence in vitro by using a method for overlapping polymerase chain reaction (PCR); directionally cloning to Pet-32c expression type carrier by two enzyme cutting sites of Nco1 and Xho1; converting the built multi-epitope peptide recombination expression vector TgMEP / Pet-32c into BL21 host bacteria; expressing soluble fusion protein with the size of 23kDa by virtue of induction; ultrasonically pulverizing the expressed bacteria and separating to obtain supernatant; and filtering the supernatant by a filtering film to perform affinity purification by using a nickel column to obtain the fusion protein TgMEP. The fusion protein TgMEP can be used for detecting bow worm disease serum IgG and IgM antibodies.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a fusion protein TgMEP and its preparation method and application. It involves producing soluble fusion protein TgMEP in Escherichia coli by means of molecular cloning and gene optimization. In addition, it involves how to screen and obtain B cell epitopes with strong immunoreactivity, how to combine B cell epitopes into multi-epitope antigens, optimize and synthesize the gene sequences corresponding to the antigens, and how to introduce the synthetic gene sequences into expression vectors. In addition, it also relates to a method for inducing the soluble expression of the TgMEP protein in Escherichia coli transformed with the fusion protein TgMEP gene, and how to purify it from the soluble supernatant of the bacteria. In addition, it also involves the establishment of an enzyme-linked immunosorbent assay using the purified recombinant fusion protein TgMEP, and the methodological e...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70G01N33/68G01N33/545
Inventor 司进戴建芳渠利利
Owner THE SECOND AFFILIATED HOSPITAL OF NANJING MEDICAL UNIV
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