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Clonorchis sinensis specific antigen VAL28 and medical application

A clonorchis sinensis, specific technology, applied in the field of immunology, to achieve the effects of good sensitivity, simple operation and high stability

Inactive Publication Date: 2021-08-03
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant reports on the preparation of immune colloidal gold test strips based on Clonorchis sinensis VAL28 as antigen to detect Clonorchis sinensis infection

Method used

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  • Clonorchis sinensis specific antigen VAL28 and medical application
  • Clonorchis sinensis specific antigen VAL28 and medical application
  • Clonorchis sinensis specific antigen VAL28 and medical application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Acquisition and Identification of Specific Antigen Gene of Clonorchis sinensis

[0033] Amplification of Clonorchis sinensis VAL28 Gene

[0034] According to the gene sequence of Clonorchis sinensis VAL28 (GenBank: MH172541.1), the specific primer sequences were designed as follows:

[0035] F: CGC GGATCC TTTGTGAAGCTGCATGATC;

[0036] R: CCC AAG CTT CTTCAAGTGGCCTGGAC (the underlined part is the restriction site, and the underlined part is the protective base). The target fragment of Clonorchis sinensis VAL28 was amplified by polymerase chain reaction. The results showed that a 513bp fragment (including the VAL28 target fragment, restriction site, and protective base) was successfully amplified. (like figure 1 shown)

[0037] Construction of pMD-18T-VAL28 cloning vector and pET-28a-VAL28 expression vector

[0038] The recombinant plasmid pMD-18T-VAL28 was identified by double enzyme digestion with BamHI and HindIII enzymes, separated and identified by 1% agaro...

Embodiment 2

[0045] Firing of colloidal gold solution

[0046] Burn the colloidal gold solution by trisodium citrate reduction method, and observe with the naked eye whether the color of the colloidal gold particle solution changes, and whether there is precipitation and aggregation. The results showed that the colloidal gold solution was wine red without aggregation and precipitation. (like Figure 7 shown)

[0047] Identification of Colloidal Gold Particles

[0048] Then, the diameter and dispersion of the colloidal gold particles can be observed by shooting a transmission electron microscope. The results show that the colloidal gold particles have a particle size of about 40nm, uniform size and uniform distribution (such as Figure 8 shown); gold particles labeled with SPA protein surrounded by a halo (as shown in Figure 9 shown).

[0049] Determination of optimum pH

[0050] Different volumes of 0.2mol / LK 2 CO 3 Add 1mL colloidal gold solution in turn, add 20μg SPA protein to...

Embodiment 3

[0069] Determination of properties of colloidal gold test strips

[0070] specificity test

[0071] Use the assembled test strips to test the positive samples of Clonorchis sinensis (a), negative samples of Clonorchis sinensis (b), positive samples of Mesorchis orientalis (c), positive samples of Fasciola hepatica (d), Toxoplasma canis The worm-positive serum (e), Neospora-positive serum (f), and Dicoelax-positive serum (g) were tested to observe whether the test line developed color. The results show that the method has good specificity (such as Figure 14 shown).

[0072] sensitivity test

[0073] Under the premise of determining the optimal coating concentration of the detection line and the quality control line, the positive serum of Clonorchis sinensis was diluted several times, and the color development of the T line of each test strip was observed to judge the sensitivity of the prepared colloidal gold test strip. The results show that the sensitivity reaches 1:200 ...

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Abstract

The invention relates to a specific antigen gene VAL28 of clonorchis sinensis and medical application thereof, and the specific antigen gene VAL28 is a novel gene protein. The colloidal gold test strip prepared on the basis of the clonorchis sinensis VAL28 protein has the advantages of high stability, strong specificity, good sensitivity, low detection cost and the like, is convenient to use, simple to operate, simple and clear in detection result and suitable for clinical rapid diagnosis, solves the problems that clinical disease detection and diagnosis time is short, the number of samples to be detected is large and the like, and has good application prospects. Meanwhile, the disease burden caused by clonorchiasis sinensis can be reduced to a certain extent through the good detection method.

Description

technical field [0001] The invention relates to a Clonorchis sinensis specific antigen gene VAL28, and a colloidal gold immunochromatographic test paper for detecting Clonorchis sinensis infection developed based on VAL28 as an antigen, which can be used for serum detection of Clonorchis sinensis infection and belongs to the technical field of immunology . Background technique [0002] Clonorchiasis, also known as liver fluke, is caused by Clonorchis sinensis ( Clonorchis sinensis ) parasitic in the liver and bile ducts of mammals including humans, is an important food-borne zoonotic parasitic disease mainly caused by liver and gallbladder lesions. The endemic area of ​​Clonorchis sinensis overlaps with the endemic area of ​​viral hepatitis, posing a major threat to global public health security. The risk of Clonorchis sinensis infection is 4.5 times that of normal people, and the risk of liver cirrhosis and liver cancer is also doubled. Therefore, serological detection a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10G01N33/558G01N33/58
CPCC07K14/43559G01N33/558G01N33/587
Inventor 李建华任妍妍张西臣宫鹏涛张楠王玉茹李新王晓岑杨举马赫然
Owner JILIN UNIV
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