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Preparation method and applications of shrimp clathrin light chain gene and targeted dsRNA thereof

A clathrin and light chain gene technology, applied in the field of clathrin light chain genes, can solve the problem that the molecular mechanism needs to be further confirmed.

Inactive Publication Date: 2014-09-24
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] For WSSV, it has been reported that the caveolae-mediated endocytic pathway may be involved in virus infection or crayfish (Duan H, Jin S, Zhang Y, et al. Granulocytes of the red claw crayfish Cherax quadricarinatus can endocytosebeads, E.coli and WSSV, but in different ways[J].Dev Comp Immunol,2014; Huang Z J,Kang ST,Leu J H,et al.Endocytic pathway is indicated for white spot syndrome virus(WSSV)entry inshrimp[J].Fish Shellfish Immunol ,2013,35(3):707-715), but its molecular mechanism needs further confirmation

Method used

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  • Preparation method and applications of shrimp clathrin light chain gene and targeted dsRNA thereof
  • Preparation method and applications of shrimp clathrin light chain gene and targeted dsRNA thereof
  • Preparation method and applications of shrimp clathrin light chain gene and targeted dsRNA thereof

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Embodiment 1

[0028] Embodiment 1 red claw crayfish Cq-CLC double-stranded RNA synthesis

[0029] refer to T7Transcription Kit design primers and prepare dsRNA.

[0030] dsDNA synthesis and purification

[0031] Take 2ng of the plasmid linked with the full length of Cq-CLC cDNA as a PCR template, and prepare the PCR reaction system as follows:

[0032]

[0033] After mixing, the PCR reaction was carried out on a thermal cycler according to the following program: 94°C, 5min; 30 cycles (94°C, 30s; 60°C, 30s; 72°C, 35s); 72°C, 3min.

[0034] PCR products were electrophoresed on 1% agarose gel to detect the amplification effect.

[0035] The PCR product of a single amplified band was purified with QIAquick PCR Purification Kit (Qiagen, Germany) to obtain double-stranded template DNA.

[0036] dsRNA synthesis and purification

[0037] Prepare the dsRNA synthesis reaction system as follows:

[0038]

[0039] After mixing, incubate in a 37°C incubator for 21-22 hours.

[0040]After t...

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Abstract

The invention provides a preparation method and applications of a shrimp clathrin light chain gene and a targeted dsRNA thereof, relating to a clathrin light chain gene. The clathrin light chain gene originates from C. quadricarinatus, and is named as Cq-CLC. The dsRNA targeted gene of the targeted shrimp clathrin light chain gene is Cq-CLC, and is named as Cq-CLC dsRNA. The preparation method comprises the following steps: 1) designing a primer and preparing dsRNA referring to a kit; 2) transfecting C. quadricarinatus Hpt cells by the dsRNA obtained in the step 1), and detecting the knock-out efficiency on the targeted gene Cq-CLC; and 3) knocking out the Cq-CLC according to the step 2), infecting WSSV, detecting endocytosis amount of WSSV in the Hpt cells knocked-out by the Cq-CLC, and comparing a control group. The Cq-CLC can be used as an important target spot of developing an anti-WSSV class new drug.

Description

technical field [0001] The present invention relates to a kind of clathrin light chain gene (Clathrin light chain, CLC), relate in particular to a kind of CLC-Cq-CLC gene that is isolated from red claw crayfish (Cherax quandricarinatus), this gene participates in the white spot virus (WSSV) of prawn ) into the cell infection; a double-stranded RNA (Double strands RNA, dsRNA) targeting Cq-CLC is also involved, and the knockout of the Cq-CLC gene mediated by the dsRNA can inhibit WSSV infection. Background technique [0002] Endocytosis is the process by which cells transport extracellular substances into the cell through the deformation of the plasma membrane. The transported substances include some macromolecular substances (such as sugars, lipids, proteins, etc.) or particles (such as bacteria, viruses). According to the difference of entry mechanism, cell endocytosis can be divided into many different ways, such as clathrin-mediated endocytosis, macropinocytosis, caveolae-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10
Inventor 刘海鹏陈荣元王克坚
Owner XIAMEN UNIV
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