Immunofluorescence reagent for detecting E-type enterovirus and detection kit thereof

An enterovirus and immunofluorescence technology, applied in the biological field, can solve the problems of endangering the healthy development of the cattle industry, lack of research on diagnosis and prevention, and achieve the effect of ensuring sensitivity, high sensitivity, and high sensitivity

Inactive Publication Date: 2016-05-04
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Type E enterovirus infection is a new infectious disease in China, which seriously endangers the healthy development of the cattle industry, and there is a lack of research on its diagnosis and prevention

Method used

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  • Immunofluorescence reagent for detecting E-type enterovirus and detection kit thereof
  • Immunofluorescence reagent for detecting E-type enterovirus and detection kit thereof
  • Immunofluorescence reagent for detecting E-type enterovirus and detection kit thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] The construction of embodiment 1E enterovirus structural protein VP1 prokaryotic expression recombinant plasmid

[0023] 1. Primer design

[0024] According to the base composition of the target sequence, primers were designed to amplify the VP1 gene, and the upstream and downstream contained BamHI and EcoRI restriction sites respectively. The primer sequences are as follows:

[0025] E-VP1-FCGC GGATCC GAAACAAGCGTGGAGA (BamHI)

[0026] E-VP1-RCCG GAATTC GTACGAGGTGAGGCT (EcoRI)

[0027] 2. Gene amplification

[0028] Genomic RNA of enterovirus E was extracted by the conventional Trizol method, and cDNA was obtained by using a commercially available conventional reverse transcription kit according to the instructions, and the VP1 gene was amplified using it as a template, and its base sequence is shown in the sequence table SEQ ID NO. As shown in 1, the amino acid sequence of expressed protein VP1 is shown in SEQ ID NO.2; PCR reaction system:

[0029]

[0030]...

Embodiment 2V

[0033] Expression and purification of embodiment 2VP1 recombinant protein

[0034] After the identified positive recombinant plasmid pGEX-4T-1-VP1 was transformed into BL21(DE3) competent cells, a single colony was picked and inoculated into 3 mL of LB liquid medium containing 100 μg / mL ampicillin for overnight culture, and then 1 mL of the above The culture was inoculated into 200 mL LB liquid medium containing 100 μg / mL ampicillin and cultured with shaking at 37 °C until the logarithmic growth phase (OD 600 =0.6~0.8), adding IPTG to a final concentration of 1mmol / L, inducing culture at 20°C for 3h, and detecting by SDS-PAGE, the recombinant target protein VP1 was obtained, as shown in figure 2 shown. Centrifuge and precipitate the bacteria, and after sonication, use urea to purify inclusion bodies to obtain high-purity recombinant proteins, such as image 3 shown.

Embodiment 3

[0035] Example 3 Preparation of anti-E enterovirus VP1 protein monoclonal antibody

[0036] 1. Animal immunization: select healthy BALB / c mice aged 6-8 weeks, emulsify the purified VP1 protein with Freund's complete adjuvant, inject about 100 μg intraperitoneally into each mouse, and emulsify the protein with Freund's incomplete adjuvant 14 days later Inject 100 μg intraperitoneally, and inject 100 μg purified protein directly intraperitoneally during the last booster immunization, and inject 50 μg purified protein into the tail vein 3 to 4 days before fusion;

[0037] 2. Cell fusion: Take splenocytes from immunized mice and mix them with SP2 / 0 in a fusion tube, centrifuge at 300g for 10 minutes, discard the supernatant, shake the cells to mix the two cells as evenly as possible, and then slowly drop and preheat within 60 seconds PEG-4000 solution, then slowly add serum-free 1640 medium to terminate the fusion, let it stand still and then centrifuge at 1000r / min for 10min, dis...

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Abstract

The invention discloses an immunofluorescence reagent for detecting an E-type enterovirus and a detection kit thereof. A direct immunofluorescence detection method set up by applying the immunofluorescence reagent for detecting the E-type enterovirus can be used for clinically fast diagnosing E-type enterovirus infection and used for positioning and authenticating an E-type enterovirus antigen in tissue. The time for direct immunofluorescence detection is short, and a result can be reported within 40 min; specificity is high, and the immunofluorescence reagent only reacts with the E-type enterovirus; the immunofluorescence reagent is high in repeatability and accuracy, the coincidence rate between results of immunofluorescence reagent and results of an indirect immunofluorescence method is 90% or more, and the immunofluorescence reagent can be used for fast diagnosing E-type enterovirus infection.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to an immunofluorescent reagent for detecting type E enteroviruses and a detection kit thereof, which are suitable for the diagnosis of type E enterovirus infection and the positioning and identification of bovine enterovirus antigens in tissues or cells. Background technique [0002] Bovine enterovirus (BEV) includes two virus species, E and F, and is a member of the genus Enterovirus in the family Picornaviridae. The infectious diseases it causes cause serious economic losses to the cattle industry. The viral infection is clinically characterized by fever, lacrimation, cough, runny nose, dyspnea, and severe diarrhea. The disease was first reported by Moll et al. in the late 1950s, and many countries have also reported the occurrence of this disease one after another. In 2011, Li Yingli et al. isolated a strain of Enterovirus F from calf feces samples from a dairy ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/577G01N33/56983G01N2333/085
Inventor 王新平邢泽黎朱利塞郭昌明刘丹盖小春张群袁悦刘亚静王方
Owner JILIN UNIV
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