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Preparation method of recombinant protein of porcine h1n1 subtype influenza virus hemagglutinin and liquid chip detection kit for the virus antibody

A technology of swine influenza virus and recombinant protein, which is applied in the liquid phase chip detection kit for the virus antibody and the preparation of swine H1N1 subtype influenza virus hemagglutinin recombinant protein, which can solve the problems of cumbersome operation, long cycle time and poor sensitivity

Active Publication Date: 2020-12-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the HI detection method is simple and easy to operate, but the sensitivity is poor; the MIVN detection method has good sensitivity and specificity, but the operation is cumbersome and the cycle is long

Method used

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  • Preparation method of recombinant protein of porcine h1n1 subtype influenza virus hemagglutinin and liquid chip detection kit for the virus antibody
  • Preparation method of recombinant protein of porcine h1n1 subtype influenza virus hemagglutinin and liquid chip detection kit for the virus antibody
  • Preparation method of recombinant protein of porcine h1n1 subtype influenza virus hemagglutinin and liquid chip detection kit for the virus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Construction of embodiment 1 expression plasmid

[0043] 1. Extraction of influenza virus total RNA and synthesis of cDNA

[0044] Refer to the instructions of the RNA extraction kit to extract the total viral RNA. After the total RNA was obtained, the reverse transcription operation was performed referring to the instruction manual of the M-MLV reverse transcriptase of Treasure Bioengineering (Dalian) Co., Ltd. The reaction system is shown in Table 1.

[0045] Table 1 Reverse transcription system

[0046] Reagent name volume RNA 9.5μL 5×MLV Buffer 4.0 μL MLV reverse transcriptase 1.0 μL dNTPs (2.5mmol each) 4.0 μL RNase Inhibitor (20U / μL) 0.5μL reverse transcription primer 1.0 μL total 20 μL

[0047] The reagents in Table 1 were added and mixed thoroughly, and placed in a water bath at 42°C for 1 hour, and the obtained cDNA product was stored at -20°C for future use.

[0048] 2. Amplification of SIV H1...

Embodiment 2

[0056] Expression, purification and identification of embodiment 2 recombinant protein

[0057] Rosetta (DE3) Escherichia coli containing the recombinant plasmid pMAL-cHA was inoculated into Luria-Bertani (LB) liquid culture fluid, and when the culture OD value reached 0.6, adding a final concentration of 0.3 mM isopropylthiogalactoside ( IPTG), after incubating at 37°C for 2 hours, the product was collected for SDS-PAGE detection; Western blot method was used to detect the expressed product with anti-SIV HA monoclonal antibody as the primary antibody. At the same time, the bacterial cells were collected by centrifugation, ultrasonically disrupted in an ice bath, and after centrifugation again, the precipitate and supernatant were tested by SDS-PAGE to analyze the solubility of the expression product.

[0058] The size of the HA target protein was in line with expectations, and there was a band at about 100KD ( image 3 ), and the target protein was expressed in both the supe...

Embodiment 3

[0064] Example 3 Application of Rosetta-pMAL-cHA to establish a liquid phase chip detection method

[0065] 1. Coupling of antigens to microspheres

[0066] Refer to Luminex's Technical encyclopedia, two-step amide reaction: first, the phosphorylated microspheres are activated by disodium hydrogen phosphate buffer, Sulfo-NHS and EDC solution, and then the HA recombinant protein prepared in Example 2 is used as an antigen to form a covalent Amide bonds, microspheres coupled with antigens were resuspended in PBS-TBN solution and stored at 4°C in the dark. The specific operation steps are as follows:

[0067] (1) Vortex the microsphere suspension for 1 min to disperse the microspheres evenly;

[0068] (2) Transfer 100 μL of microspheres to a 1.5mL centrifuge tube, centrifuge at 8000g / min for 2min, place on a magnetic stand, and gently remove the supernatant (do not suck away the microspheres);

[0069] (3) Add 100μL ddH 2 O, vortex for 1min, then centrifuge at 8000g / min for...

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Abstract

The invention first provides a preparation method of a swine H1N1 subtype influenza virus hemagglutinin (HA) recombinant protein. A pair of primers with HIS labels is designed by removing a signal peptide of the HA according to an HA sequence, and a prokaryotic expression plasmid with a 6*His-maltose-binding protein (MBP) combination label is constructed; the HA recombinant protein expressed in a soluble form is successfully obtained in an Escherichia coli expression system and can generate reaction with a mouse antibody with swine H1N1 subtype influenza virus HA, thereby showing that the HA recombinant protein has good antigenicity; a liquid phase chip detection technology is established by using the HA recombinant protein and is higher than ELISA (Enzyme-Linked Immuno Sorbent Assay) in sensitivity; the establishment of the method provides necessary technical supplement for detecting a swine influenza virus antibody, lays a technical basis for researching the liquid phase chip detection technologies of other epidemic disease antibodies, and provides a test reference for establishing multiple liquid phase chip detection technologies of multiple swine disease antibodies.

Description

technical field [0001] The invention relates to the technical field of virus detection, and more specifically, relates to a method for preparing a porcine H1N1 subtype influenza virus hemagglutinin recombinant protein and a liquid phase chip detection kit for the virus antibody. Background technique [0002] Swine Influenza (SI) is an acute, febrile, highly contagious respiratory infectious disease caused by Swine Influenza virus (SIV). , high morbidity and low mortality. At present, the classic swine influenza H1N1 and human-like H3N2 subtypes are widely prevalent in swine populations around the world. Although the mortality rate of swine influenza is low, it can cause secondary infection of other pathogens and cause huge economic losses to the breeding industry. Pigs are susceptible hosts for human and avian influenza viruses, intermediate hosts for cross-species transmission of influenza viruses, "mixers" for genetic recombination of influenza viruses, and an important c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/11C12N15/70G01N33/569G01N33/543C09K11/06C12R1/19
CPCC07K14/005C09K11/06C09K2211/14C12N15/70C12N2760/16122C12N2800/101G01N33/54313G01N33/56983
Inventor 张桂红王衡记方晓孙彦阔冀池海曾梦
Owner SOUTH CHINA AGRI UNIV
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