Method for inducing blood lymphocyte of litopenaeus vannamei to generate UPR (unfolded protein response) by means of RNA interference technology

An RNA interference and blood lymphocyte technology, applied in the field of molecular biology RNA interference technology and application, can solve the problem of not finding specific activation of UPR and so on

Inactive Publication Date: 2015-03-25
SUN YAT SEN UNIV
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Problems solved by technology

To study the relationship between UPR and immune system of Litopenaeus vannamei, the relationship between UPR and virus infection, the relationship between UPR and cell cycle regulation, and the relationship between UPR and substance metabolism requires an effective and fast method for inducing UPR in Litopenaeus vannamei cells. However, a method to specifically activate the UPR in Litopenaeus vannamei has not been found so far

Method used

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  • Method for inducing blood lymphocyte of litopenaeus vannamei to generate UPR (unfolded protein response) by means of RNA interference technology
  • Method for inducing blood lymphocyte of litopenaeus vannamei to generate UPR (unfolded protein response) by means of RNA interference technology
  • Method for inducing blood lymphocyte of litopenaeus vannamei to generate UPR (unfolded protein response) by means of RNA interference technology

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Embodiment

[0038] First, design and synthesize the primers for dsLvBip's forward ssRNA and reverse ssRNA templates, synthesize dsLvBip's ssRNA and reverse ssRNA through PCR reaction, and then form dsLvBip through denaturation and annealing. Inject dsLvBip into the second abdominal segment of Litopenaeus vannamei at a dose of 1μg / g body weight. 48-72 hours after injection, a sample of Litopenaeus vannamei blood lymphocytes was taken to extract total RNA, reverse transcribed into cDNA, and then the interference effect of total Bip gene (GenBank under accession No.JQ265942) and UPR activation of Litopenaeus vannamei blood lymphocytes were tested effect.

[0039] Design two pairs of primers to synthesize ssRNA and reverse ssRNA templates:

[0040] DsRNA-LvBip-482-T7-F1:

[0041] GGATCCTAATACGACTCACTATAGGTGTCGGTGGTTCCACTCGTA

[0042] DsRNA-LvBip-482-R1:CCTTGTTTCCTGTGCCCTTG

[0043] DsRNA-LvBip-482-F2:TGTCGGTGGTTCCACTCGTA

[0044] DsRNA-LvBip-482-T7-R2:

[0045] GGATCCTAATACGACTCACTATAGGCCTTGTTTCCTGTGC...

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Abstract

The invention provides a method for inducing blood lymphocyte of litopenaeus vannamei to generate UPR (unfolded protein response) by means of an RNA interference technology. The method comprises the following steps: (1) synthesizing dsLvBip with sequence specificity according to a cDNA sequence of a Bip gene of litopenaeus vannamei; and (2) diluting dsLvBip, controlling the injection volume at 50 mu L and injecting dsLvBip into the litopenaeus vannamei according a dosage of 1 mu g / g. The method provided by the invention can be used for simply, conveniently, rapidly, efficiently and specifically inducing blood lymphocyte of litopenaeus vannamei to generate UPR.

Description

Technical field [0001] The invention belongs to the field of molecular biology RNA interference technology and application, and specifically relates to a method for using RNA interference technology to knock down the Bip gene of Litopenaeus vannamei to induce UPR in blood lymphocytes. Background technique [0002] The prawn (Litopenaeus vannamei) in the marine invertebrate genus Penaeus is an important species of aquaculture in my country. In 2013, the global production of farmed shrimp reached 3.3 million tons, of which my country's production of farmed shrimp was 1.3 million tons, making it the world's largest shrimp farming country. my country mainly cultivates four species of prawns, Litopenaeus vannamei, Penaeus monodon, Fenneropenaeus chinensis and Marsupenaeus japonicus. Among them, Litopenaeus vannamei is the main cultured species. The 11 coastal provinces and cities across the country have farmed, accounting for about 90% of the total farmed shrimp production. [0003] Wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/89
Inventor 陈义烘张泽芝翁少萍何建国
Owner SUN YAT SEN UNIV
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