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Application of Aedes albopictus salivary 34k2 recombinant protein in hacat cells infected by denv2

A technology of recombinant protein and Aedes albopictus, applied in the biological field, can solve problems such as unclear functions and achieve the effect of promoting diffusion

Active Publication Date: 2020-12-04
GUIZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our previous research found that mosquito blood sucking can regulate the expression of 34K2 protein, but the role of this protein in mosquito blood sucking and disease transmission is not clear

Method used

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  • Application of Aedes albopictus salivary 34k2 recombinant protein in hacat cells infected by denv2
  • Application of Aedes albopictus salivary 34k2 recombinant protein in hacat cells infected by denv2
  • Application of Aedes albopictus salivary 34k2 recombinant protein in hacat cells infected by denv2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Analysis of rAb-34K2 protein on the proliferation of HaCaT cells

[0037] In order to detect whether different concentrations of rAb-34K2 protein have a direct effect on the proliferation of HaCaT cells, in this experiment, the rAb-34K2 salivary protein was diluted with Hepes buffer and divided into 1 μg / mL, 5 μg / mL, and 10 μg / mL groups. Set the BSA protein control, negative control (Hepes buffer control) and blank control with corresponding concentration gradients, inoculate the E-Plate detection plate with the number of human keratinocytes (HaCaT) at 2×104 cells / 100 μL, and use RTCA to dynamically monitor the cells Changes in the growth curve, to detect the effect of different concentrations of rAb-34K2 saliva protein on the growth of HaCaT cells.

[0038] The above-mentioned RTCA method for dynamically monitoring the cell growth curve is as follows:

[0039] (1) Add DMEM to the E-Plate16 plate for complete culture and detect the baseline based on RTCAStations;

[0...

Embodiment 2

[0049] Analysis of the function of rAb-34K2 protein on virus proliferation in DENV2-infected HaCaT cells

[0050] (1) The effect of rAb-34K2 protein on the cytopathic effect (CPE) of DENV2 infected HaCaT cells:

[0051] Proliferation and identification of DENV2: Dilute DENV2NGC strain virus 50 times, inoculate C6 / 36 cells in logarithmic growth phase, and set the cells not inoculated with virus as negative control. 37°C, 5% CO 2 Incubate in an incubator. After the cells have obvious lesions (swelling, fusion, vacuoles, etc.), freeze and thaw them repeatedly 3 times, centrifuge at 4000 rpm for 5 minutes to collect the supernatant, obtain DENV2 virus liquid and use primer NS1F (SEQ ID NO: 1 ) and NS1R (SEQ ID NO: 2) to perform RT-PCR amplification on the NS1 gene fragment, the reaction system of the RT-PCR amplification is shown in Table 2, and the reaction program is 95°C, 8min; 95°C, 30S; 60°C, 30S; 72°C extension for 30S, 35 cycles, and 5 μL of PCR amplification product was ...

Embodiment 3

[0074] Analysis of regulation of cytokine expression by rAb-34K2 protein on DENV2-infected HaCaT cells

[0075] (1) The effect of rAb-34K2 salivary protein on the expression of type I interferon in DENV2 infected HaCaT cells IFN-α / β gene detection standard preparation method is the same as ① in (2) in Example 2, and the preparation of the standard curve is the same as in the example ② in (2) in 2, the detection of nucleic acid is the same as ③ in embodiment 2 (2).

[0076] Table 7 The relative expression of IFN-α and IFN-βmRNA after different concentrations of rAb-34K2 protein and DENV2 (MOI=1) acted on HaCaT cells

[0077]

[0078]

[0079] This experiment quantitatively detected the relative expression of IFN-α / β mRNA in each infection group at infection 12h and 24h when the MOI of DENV2 infection was 1. The difference between the groups was not obvious; while the expression of each group was up-regulated at 24 hours after infection, but the relative expression of t...

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Abstract

The invention relates to the technical field of biology, in particular to an application of a recombinant aedes albopictus salivary 34K2 (short for rAb-34K2DENV2) protein to promotion of DENV (Dengue virus) multiplication. A preparation method comprises the step that the rAb-34K2DENV2 protein can promote multiplication of DENV2 in HaCaT cells. The mechanism is that theDENV2-infectedHaCaT cells are inhibited from expressing type-I interferon IFN-alpha / beta to promote virus multiplication, meanwhile, the rAb-34K2protein can induce apoptosis of the DENV2-infectedHaCaTcells, and great significance in study of the function of aedes albopictus salivary protein components in a disease transmission mechanism of mosquitos is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of Aedes albopictus saliva 34K2 recombinant protein in DENV2 infection of HaCaT cells. Background technique [0002] Mosquito saliva protein has the functions of anticoagulation, anti-inflammation and immune regulation, and is a hotspot of current research. Salivary gland transcriptomics and proteomics studies have found that in the salivary glands of Aedes mosquitoes, there is a group of salivary component proteins with a molecular weight of 34kDa and unknown functions that are specifically highly expressed in female mosquitoes, so they are named 34K protein family. The protein coding genes all contain signal peptide sequences, indicating that they are involved in the composition of mosquito saliva component proteins. Aedes albopictus, commonly known as the mosquito mosquito, is also known as the "Asian tiger mosquito" because of its ferocious nature and strong atta...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N7/00C12R1/93
CPCC07K14/43563C12N7/00C12N2770/24151
Inventor 吴家红商正玲程金芝马亚萍
Owner GUIZHOU MEDICAL UNIV
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