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ALV-K ELISA kit and detection method thereof

A kit, the technology of his-k-gp85, applied in the directions of microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as the advent, the absence of vaccines and therapeutic drugs, the difficulty of purification of avian leukemia, etc. To achieve the effect of optimizing reaction conditions and improving reliability

Active Publication Date: 2020-04-10
YANGTZE UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented describes various methods for producing high levels of certain proteins called alveolar macrophage (AM) cellular receptors or humoral factors like IL-33 produced during inflammatory responses associated with sepsis syndrome. These AMs have been shown to play key roles in regulating innate defense mechanisms such as neutrophy induction, complement activation, cytokine production, and chemotaxis toward targeting molecules involved in pathogenesis. By combining these components together, they may provide new treatments against bacterial diseases caused by Gram-negative staphylococci.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving the effectiveness and reliance of current methods used for detecting or prevention against avians Leukemia Virus (AvLv), specifically monocyte lymphoma viruses (MLSV)-related diseases such as Chikungunya fevers (CLCFUs)).

Method used

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  • ALV-K ELISA kit and detection method thereof
  • ALV-K ELISA kit and detection method thereof
  • ALV-K ELISA kit and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Example 1 Construction of recombinant expression vector

[0056] 1. gp85 gene amplification

[0057] According to the gp85 sequence (as shown in SEQ ID NO.1) of published ALV-K strain in GenBank, design a pair of primers that can specifically amplify the gp85 gene, respectively in the forward primer gp85-F (as shown in SEQ ID NO. .2) and the reverse primer gp85-R (shown in SEQ ID NO.3) were introduced into the respective 5' ends of BamHI and SalI restriction sites, as shown in Table 1.

[0058] Table 1 gp85 gene-specific amplification primers

[0059] Primer sequence TM value / ℃ gp85-F 5'-CGC GGATCC CACTTACTCGAGCAGCCAGGGAAC-3'

70.8 gp85-R 5'-ACGC GTC GAC GGGTGCTTCGTTTACGTCTTATACC-3'

67.3

[0060] Using the genomic DNA of DF-1 cells infected with the ALV-K strain as a template, and using gp85-F and gp85-R as primers, the gp85 gene was amplified by PCR. The PCR used a 25 μL reaction system. The system is shown in Table 2:

...

Embodiment 2

[0084] Example 2 Induction and expression of recombinant HIS-K-gp85 protein

[0085] The recombinant expression vector pET-28a-ALV-K-gp85 obtained in Example 1 was transformed into the E.coliBL21 expression strain by the heat shock method, and evenly spread on the Amp and Kana plates after recovery, and after culturing overnight at 37°C, After the positive colonies were selected and identified, the correct colonies were inserted into two liquid LB medium (4 mL each) filled with corresponding antibiotics to shake the bacteria, one of which was used for subsequent experiments and the other was used for induction. When the OD value of the bacterial solution is about 0.6, take out 3 sterilized clean test tubes, add 1mL of the bacterial solution and 1μL of 1mol / L IPTG to each test tube until the medium concentration is 1mM, as the induction group; the remaining bacteria in the original test tube solution as the uninduced group; the induced group set a temperature gradient of 16°C...

Embodiment 3

[0086] Example 3 Purification of recombinant HIS-K-gp85 protein

[0087] Wash the collected E.coliBL21 cells with 30mL Buffer A at 12000r / min; refrigerate and centrifuge at 4°C for 10min, discard the supernatant; resuspend the cells with Buffer C, add lysozyme and protease inhibitor (PMSF) until Concentrations are 1mg / mL and 1mM respectively, ice bath in ice-water mixture for half an hour, and then put the resuspended bacterial solution in the ultrasonic breaker to lyse, break for 5s, interval 10s, power 200W, total working time 90min; end of ultrasonic lysis Finally, 12000r / min; refrigerated centrifugation at 4°C for 20min, filtered through a 0.45μm filter to remove impurities, and the protein solution to be purified was obtained for future use.

[0088]Rinse the empty column of gravity chromatography with deionized water repeatedly for 3 times, add 3mL NiFocurose6FF to the empty column after washing, wash Ni Focusose 6FF twice in the column with PBS, then wash twice with B...

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Abstract

The invention discloses an ALV-K ELISA detection kit and a detection method thereof. A coding sequence of the protein is as shown in SEQ ID NO. 1. The method comprises the following steps: cloning a gene encoded with SEQ ID NO.1 to an expression vector to obtain a recombinant expression vector; and converting the recombinant expression vector into a host cell for expression and purification to obtain the recombinant HIS-K-gp85 protein. A rabbit polyclonal antibody KE7 is obtained by immunizing a large ear rabbit with the recombinant HIS-K-gp85 protein as an antigen, and the ALV-K ELISA detection kit and the detection method thereof are established. According to the invention, the recombinant expression vector pET-28a-ALV-K-gp85 is successfully constructed through recombination of a gp85 gene, and a soluble recombinant HIS-K-gp85 protein is obtained; and the ALV-K ELISA detection method established by the invention has the advantages of high credibility, strong specificity, high sensitivity and good repeatability.

Description

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Claims

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Application Information

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Owner YANGTZE UNIVERSITY
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