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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  • Abstract
  • Description
  • Claims
  • Application Information

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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

Method used

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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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Example Embodiment

[0020] Example 1: Expression, purification and immunoreactivity detection of Toxoplasma 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, the six common diagnostic antigen molecules of Toxoplasma gondii, the main surface antigen 1 (SAG1), the main Analysis of B cell epitopes of surface antigen 2 (SAG2), major surface antigen 3 (SAG3), tachyzoite surface antigen P35, dense granule protein 1 (GRA1), and dense granule protein 6 (GRA6); each antigen molecule is selected Two predicted B cell epitopes, two complementary single-stranded oligonucleotides were designed according to the sequence of each epitope, annealed to form a double strand and cloned into the...

Example Embodiment

[0032] Example 2 Application of Recombinant Fusion Protein TgMEP in Toxoplasmosis Diagnostic Kit

[0033] [1] Establishment of enzyme-linked immunosorbent assay of recombinant fusion protein TgMEP

[0034] Orthogonal experiments were used to determine the optimal detection conditions of the enzyme-linked immunosorbent assay for the detection of toxoplasmosis IgG and IgM antibodies. The coating concentrations of the recombinant fusion protein TgMEP for the detection of IgG and IgM antibodies were: 1μg / ml. And 2μg / ml. The diagnostic cut-off value was the mean value of 15 negative sera plus 2 standard deviations. The diagnostic cut-off values ​​of the enzyme-linked immunosorbent assay for the detection of IgG and IgM antibodies were 0.14 and 0.16, respectively. The specific detection steps are: coating polystyrene enzyme label strip with 100μl / well of carbonic acid buffer containing recombinant fusion protein, overnight at 4℃, blocking with 5% skimmed milk powder in PBST at 37℃ for 1...

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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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