Toxoplasma gondii ROP17 recombinant protein as well as preparation method and application thereof

A recombinant protein, Toxoplasma gondii technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., to achieve the effects of high sensitivity, good antigenicity, and high sensitivity detection

Pending Publication Date: 2022-06-10
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, scholars at home and abroad mostly use Toxoplasma gondii tachyzoites cultured by tissue or cells as antigens. Although this method has good specificity, there are problems such as uncertain antigen composition, low purity, and large differences between antigen preparation batches.

Method used

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  • Toxoplasma gondii ROP17 recombinant protein as well as preparation method and application thereof
  • Toxoplasma gondii ROP17 recombinant protein as well as preparation method and application thereof
  • Toxoplasma gondii ROP17 recombinant protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 end-28a-Rop17 Embarrants The Construction of Early Cerfinal Expressive Version

[0049] 1. Primer design of the Rop17 gene

[0050] Design primers based on the Gow17 gene sequence (NCBI login number is AM075203.1), and the 5 ' -end connection enzyme cutting point of the upstream and downstream primers BAMHI and XHOI, the corresponding base protection (BAMHI and XHOI, and the alkaline base baseSee the lower line of the primer sequence).

[0051] The sequence of the above primers is specifically:

[0052] Upstream primer f (Rop17-F): 5’- Cgggatcc Atggggttgtgtgtgcttttg-3 (seq ID No.1);

[0053] Downstream primer R (Rop17-R): 5’- CCGCTCGAG CTCCTCTGTGTAAAAGCCCCCCT-3 (seq IDNO.2).

[0054] 2. PCR amplification ROP17 gene

[0055] The bow -shaped worm Rh plant speed colonial is the liquid nitrogen frozen in this laboratory (given by Professor Zhu Xingquan of the Lanzhou Veterinary Institute of the Chinese Academy of Agricultural Sciences), and uses Vero cells to be train...

Embodiment 2

[0072] Example 2 Rop17 Reorganization protein expression and purification

[0073] 1. PET28A (+) -The induction expression of Rop17 recombinant plasmid

[0074] (1) It will be transformed to BL21 (DE3) monoclonal inoculation of 5ml with KAN + In the LB liquid medium, the cultivation of about 7h, then inocus at a 150ml LB medium in a 1:00 ratio for expansion and culture, placed in a 37 ° C shock culture box, and 180rpm shock training.

[0075] (2) When growing to the right period (OD 600 At about 0.6 pm), the IPTG with a final concentration of 1mmol / L is induced, and the PET28A (+) empty carrier-induced group and PET28A (+) -s Rop17 reorganized carrier did not induce the bacterial solution control group.The bacteria liquid after 8H 12000rpm centrifuge 5min, collect the precipitation of the bacteria, wash it with PBS once, and add the PBS heavy suspension (per ML bacteria required about 5ml of PBS).For 200W, 10S each time, 10s interval, 4 times in a total of 4 ° C, 12000rpm centrifu...

Embodiment 3

[0099] Example 3 Toxoplasma Rop17 protein immune mice and serum preparation

[0100] Purified ROP17 reorganization of the Erotic Expressive expression of protein and equivalent FA -Sascum Emulsion, as a multi -point immune to 5 BALB / C mice under the antigen, 100 μg / piece, immune every 2 weeks, 3 times of immunity, and finally 3 times. FinallyAt one time, it uses an incomplete agent emulsifying immunity.After the end of the immune, the heart is blood collection, isolation of serum, and detecting serum cost with ELISA.The cost of the antibody of the Rop17 immune mice by ELISA method is 1: 128000, indicating that the purified Rop17 reorganization expression protein has a good specificity.

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a toxoplasma gondii ROP17 recombinant protein and a preparation method and application thereof.The preparation method includes the steps that specific primer design is conducted on a toxoplasma gondii ROP17 gene, then a ROP17 segment is amplified with toxoplasma gondii DNA as a template, the ROP17 segment is connected to a pET-28a (+) expression vector after double enzyme digestion and converted to a clone competent cell, and the recombinant protein is obtained. After plasmid is extracted, prokaryotic expression competent cells are converted to construct a prokaryotic expression vector, and the constructed prokaryotic expression vector is induced and purified to prepare the toxoplasma gondii ROP17 recombinant protein. The fusion protein ROP17 has good antigenicity, can efficiently capture target molecules in a high-density mode, achieves the purpose of rapid and high-sensitivity detection of toxoplasma gondii serum, can be suitable for indirect ELISA, sandwich ELISA and other detection methods, can accurately and rapidly detect toxoplasma gondii infection, and is high in sensitivity and specificity.

Description

Technical field [0001] The present invention is a genetic engineering technology field, which involves a reorganized protein and its preparation methods and applications in a bowl -shaped worm Rop17. Background technique [0002] Gow -shaped worm is a specialty cell parasitic that can infect all warm blood animals, including humans, has a complex life history and pathogenic mechanism.Toxoplasma insects may be transmitted to the fetus vertically, or the infectious ovulation sacs are intake from the environment through the horizontal transmission pathway or the tissue caps or instant colonials contained in different animals.Transplanted or unprecedented milk contained in milk.Due to no special effects of drugs and vaccines, bow -shaped insect diseases seriously endanger human health and animal husbandry production. [0003] Gow -shaped hormone metabolic secretion antigens mainly include microfin protein, dense granulin, and stick -like protein.Among them, stick -like protein is a b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/45C12N15/30C12N15/70C12N15/66G01N33/569G01N33/543
CPCC07K14/45C12N15/70G01N33/56905G01N33/543G01N2333/45
Inventor 张翩袁子国王晓虎张秀香陈晶任照文王刚
Owner SOUTH CHINA AGRI UNIV
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