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Eukaryotic expression CFP10-ESAT-6 fusion protein for diagnosis of bovine tuberculosis

A CFP10-ESAT-6, fusion protein technology, applied in microorganism-based methods, hybrid peptides, recombinant DNA technology, etc., can solve problems such as lack of protein phosphorylation modification processing mechanism, and achieve improved sensitivity and specificity, Overcome Escherichia coli system, fast growth effect

Inactive Publication Date: 2013-07-17
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the existing studies have used the Escherichia coli prokaryotic expression system to express, which lacks the phosphorylation, glycosylation and other modification processing mechanisms required for many proteins after translation.

Method used

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  • Eukaryotic expression CFP10-ESAT-6 fusion protein for diagnosis of bovine tuberculosis
  • Eukaryotic expression CFP10-ESAT-6 fusion protein for diagnosis of bovine tuberculosis
  • Eukaryotic expression CFP10-ESAT-6 fusion protein for diagnosis of bovine tuberculosis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Construction of eukaryotic expression engineering bacteria

[0025] 1. Primer design and target gene amplification

[0026] According to the cfp10-esat6 fusion gene sequence SEQ ID NO.4 constructed and preserved in our laboratory, primers were designed in combination with the characteristics of the pPICZαA vector (Invitrogen). The start and stop codons of the cfp10-esat6 fusion gene were removed to allow the expression of the alpha signal peptide upstream of the multiple cloning site and the downstream tag. The primer sequences are as follows:

[0027] P1 (upstream primer): 5'-GCGT GCAGAGATGAAGACCGATG-3' (SEQ ID NO.2) (the base in the box is the restriction endonuclease EcoRI recognition site);

[0028] P2 (downstream primer): 5'-TAAT TGCGAACATCCCAGTGACG-3' (SEQ ID NO.3) (the underlined base in the box is the recognition site of restriction endonuclease XbaI, and two bases AA are added to prevent c-myc and His from frameshifting).

[0029]The plasmi...

Embodiment 2

[0038] Example 2: Expression, purification and identification of recombinant proteins

[0039] 1. Induced expression of target protein

[0040] Firstly, through the exploration of induction conditions, it was determined that the optimal induction time was 3 days, and the optimal induction concentration of methanol was 1%.

[0041] Inoculate positive clones into 25mL BMGY liquid medium, culture at 28-30°C, shake at 250rpm until OD 600 =2, centrifuge at 1500g room temperature for 5min, discard the supernatant, and resuspend the cells to OD with BMMY 600 =1, the obtained bacterial solution was placed in a 1L Erlenmeyer flask, 28-30°C, 250rpm shaking culture, adding methanol to 1.0% concentration every 24h, until the 3rd day, centrifuged at the maximum speed, and separated the supernatant of the sample.

[0042] 2. Purification of target protein

[0043] After the above supernatant was concentrated, it was purified by His Bind purification kit. The specific operation was carrie...

Embodiment 3

[0045] Embodiment 3: CFP10-ESAT-6 protein is used for bovine tuberculosis intradermal allergy test

[0046] Firstly, a comparative allergy test is carried out, according to the OIE bovine tuberculosis detection comparative allergy test operation: Inject bovine PPD and avian PPD respectively on the same side of the cow's neck at an interval of about 12-15cm, measure the skin thickness of the injection site before injection, and inject After 72 hours, the skin thickness at the same location was measured again, and the difference between the two measurements was calculated. If the skin thickness difference stimulated by bovine PPD is larger than that stimulated by poultry PPD and exceeds 4mm, it is judged as positive; if the skin thickness difference is between 1-4mm, it is judged suspicious; otherwise, it is judged as negative.

[0047] The intradermal allergy test of the target protein is the same as the OIE bovine tuberculosis detection intradermal allergy test, only the eukar...

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Abstract

The invention provides a eukaryotic expression CFP10-ESAT-6 fusion protein for diagnosis of bovine tuberculosis, which is applied to clinical diagnosis of the bovine tuberculosis. A cfp10-esat6 fusion gene is obtained by PCR (polymerase chain reaction) amplification, a recombinant plasmid pPICZ alpha A-(cfp10-esat6) is constructed, the transfer into Pichia pastoris GS115 is performed, and the high-purity CFP10-ESAT-6 fusion protein is obtained by methanol induction and affinity chromatography. The eukaryotic expression CFP10-ESAT-6 fusion protein is used for an intradermal allergic reaction which is used for diagnosis of the bovine tuberculosis and an antigen-specific bovine IFN-gamma test as a stimulus, the former has the sensitivity of 47.4% and the specificity of 83.3% in comparison with the results of a comparison allergic reaction, and the later has the positive coincidence rate of 82.8% and the negative coincidence rate of 87.4 in comparison with a bovine IFN-gamma kit, thereby showing greater value in diagnosis of the bovine tuberculosis.

Description

technical field [0001] The invention relates to a mycobacterium tuberculosis CFP10-ESAT-6 fusion protein, which is obtained by eukaryotic expression in a yeast expression system and has good application prospects in the diagnosis of bovine tuberculosis. Background technique [0002] Bovine tuberculosis is a chronic bacterial disease mainly caused by Mycobacterium bovis infecting cattle. It can also infect humans and some other animals and cause corresponding diseases. It is listed as an animal disease that needs to be notified by the International Veterinary Office (OIE). Bovine tuberculosis directly affects the productivity of cattle and the international trade of products, causing huge economic losses. At the same time, as an important zoonotic disease, it is also a major threat to public health. Although the prevalence of bovine tuberculosis has been reduced to a very low level in most developed countries due to the "quarantine-cull" eradication strategy and strong econom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/81G01N33/68C12R1/84
Inventor 焦新安陈祥孟闯时振华徐正中殷月兰孙林
Owner YANGZHOU UNIV
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