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Composite antigen for detecting haemonchus contortus infection and application thereof

A technique of twisting Haemonchus contortus and compound antigens, which is applied in the fields of veterinary immunology and molecular biology, can solve the problems of high cost, killing animals, and difficulty in popularization, and achieve the effect of short time required and easy operation

Active Publication Date: 2021-11-12
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Stool examination is currently the most widely used method, but it takes about 3 weeks after infection to detect eggs in feces, which is difficult to diagnose early infection
Necropsy method can make accurate diagnosis, but it needs to kill animals, which is very expensive and difficult to promote

Method used

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  • Composite antigen for detecting haemonchus contortus infection and application thereof
  • Composite antigen for detecting haemonchus contortus infection and application thereof
  • Composite antigen for detecting haemonchus contortus infection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0081] The Western blot analysis of embodiment 2 composite protein Hc-5C

[0082] 2.1 Preparation of serum from goats infected with Haemonchus contortus at different stages

[0083]Four local goats aged 4-6 months were purchased from a farm in Jurong City, Jiangsu Province, and were bred in the animal room of Nanjing Agricultural University. After deworming, fecal examination was used to confirm that there was no parasitic infection, and artificially infect the third-stage larvae of Haemonchus contortus (L3, 5000 / only). Blood was collected before infection (0 day, negative control) and 7, 14, 21, 28, 35, 42, 49, 56, 61 days after infection and serum was separated.

[0084] 2.2 Western blot analysis of recombinant protein

[0085] Sera from different stages of goat infection were used as the primary antibody, and rabbit anti-goat HRP-IgG was used as the secondary antibody to analyze the Hc-5C recombinant protein by Western blot. result( Figure 7 ) found that Hc-5C could re...

Embodiment 3

[0088] Embodiment 3 establishes colloidal gold test strip detection technology based on recombinant Hc-5C protein

[0089] 3.1 Assembly of colloidal gold immunochromatographic test strips

[0090] (1) Soak the gold label pad in the colloidal gold-labeled antigen solution for 10 minutes, then dry it in an oven at 37°C for 30 minutes, repeat 3 times, finally cut it into a suitable size, and store it in a dry place at 4°C for later use.

[0091] (2) Prepare T-line and C-line on the NC film with a scriber at 1 μL / cm. After drying in an oven at 37°C for 30 minutes, cut it to a suitable size with a strip cutter.

[0092] (3) Paste the serum pad, NC membrane, and absorbent paper on the bottom plate in sequence.

[0093] (4) The above-mentioned materials after cutting according to Figure 8 Install and press in sequence, and store in a dry place at 4°C for later use.

[0094] 3.2 Colloidal gold labeled Hc-5C antigen

[0095] (1) Take 10mL colloidal gold solution to a 50mL centrifu...

Embodiment 4

[0118] The clinical application of embodiment 4 diagnostic method

[0119] Serum samples and abomasum samples were collected from 19 goats. Adult counts and colloidal gold dipstick tests were performed on these samples to compare the two diagnostic methods. Result (table 3) finds that colloidal gold test strip detection technology detects 14 positive samples altogether, and the detection rate is 73.4% (14 / 19), and the coincidence rate of this method and autopsy result is 84.2% ((14+ 2) / 19).

[0120] Table 3 Comparison of detection technology based on Hc-5C colloidal gold and autopsy results

[0121]

[0122] Serum and fecal samples were collected from 35 goats. Egg counts and colloidal gold test strips were performed on these samples. The results (Table 4) found that 35 positive sera were detected by the colloidal gold test strip detection technique, the detection rate was 100% (35 / 35), and the detection rate was higher than that of the fecal examination method. The co...

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Abstract

The invention relates to a composite antigen for detecting haemonchus contortus infection and application thereof. The compound antigen can be used for diagnosing the early infection of the parasites, and the amino acid sequence of the compound antigen is shown as SEQ ID NO.1. The antigen can perform specific recognition on an antibody in host serum 7 days after being infected with haemonchus contortus, and diagnosis can be performed 2 weeks before the host discharges eggs. A colloidal gold test strip detection method is established by utilizing the antigen, has the advantages of good sensitivity, strong specificity, simplicity and convenience in operation, short time and the like, and has a relatively good application prospect in prevention and control of haemonchus contortus diseases of ruminants such as cattle, sheep, deer, camel and the like.

Description

technical field [0001] The invention belongs to the technical field of veterinary immunology and molecular biology, and relates to a serological diagnosis method for animal parasitic diseases. Background technique [0002] Haemonchus contortus is a common digestive tract parasite in ruminants such as cattle, sheep, deer, and camels. It lives in the fourth stomach of the host and feeds on the blood of the host. Symptoms such as anemia, emaciation, edema, and diarrhea appear after animal infection, which can cause a large number of deaths of young animals, especially lambs. The mortality rate of adult animals is relatively low, but the growth is slow after infection, and the feed remuneration is reduced, causing huge economic losses and often not be found. With the vigorous development of cattle and sheep and other herbivorous animals and related industries in my country, the harm of Haemonchus contortus and the economic losses caused by it are becoming more and more obvious ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/558G01N33/543G01N33/53C07K14/435C07K16/18C07K16/06C12N15/12C12N15/70
CPCG01N33/6893G01N33/558G01N33/54346G01N33/5308C07K14/4354C07K16/18C07K16/065C12N15/70C12N2800/101G01N2333/4353
Inventor 严若峰李祥瑞徐立新宋小凯赖旭东
Owner NANJING AGRICULTURAL UNIVERSITY
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