Procambarus clarkia inhibin 1 gene as well as coded inhibin1 protein and application thereof

A technology of Procambarus clarkii and inhibitin, which is applied in the field of genetic engineering, and can solve problems such as the prohibitin gene and its application that have not been seen

Inactive Publication Date: 2013-09-25
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Typical representatives in shrimp, such as anti-lipopolysaccharide factor (ALF), crustins, etc., but in related reports, there is no report on the gene and application of prohibitin

Method used

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  • Procambarus clarkia inhibin 1 gene as well as coded inhibin1 protein and application thereof
  • Procambarus clarkia inhibin 1 gene as well as coded inhibin1 protein and application thereof
  • Procambarus clarkia inhibin 1 gene as well as coded inhibin1 protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Cloning of the cDNA of Procambarus clarkii PHB1

[0057] 1) Extraction of total RNA: Total RNA was extracted using the existing technology one-step method (Trizol).

[0058] 2) cDNA first-strand synthesis: 5 microliters of total RNA, plus 1 microliter of SmartF (5'-TAC GGC TGC GAG AAG ACG ACA GAA GGG-3') and 1 microliter of Oligoanchor R (5'-GAC CAC GCG TAT CGA TGT CGA CT 16 (A / C / G)-3'), react at 72°C for 5 minutes, then add 5 times Buffer 4 microliters, dNTP2 microliters, RNase inhibitor 0.5 microliters, 1 microliter M-MLV reverse transcriptase, no 6.5 microliters of RNase-sterilized water were reacted at 42°C for 60 minutes, and the reaction was terminated at 70°C for 5 minutes.

[0059] 3) Amplification of the full-length cDNA of Procambarus clarkii PHB1

[0060] According to the conserved sequence of PHB1 in other species, the degenerate primer F1 was designed, and PHB1 was amplified by PCR with the universal back primer 3'primer:

[0061] Forward pri...

Embodiment 2

[0070] Example 2: Construction, expression and purification of prokaryotic recombinant expression vectors

[0071] (1) According to the sequence of Procambarus clarkii PHB1 and the cloning site of the expression vector pET30a (Novagen Company), design primers:

[0072] FcUbcExF:5ˊTAC TCA GAA TTC ATG GGG CAG ATT GGC TTC GGT3′(EcoR I)

[0073] FcUbc ExR:5ˊTAC TCA CTC GAG TCA CTG AGG AAG AGA TAA G3'(Xho I) (as shown in SEQ ID NO.5, 6)

[0074] When the primers are designed in the present invention, an EcoR I restriction site is introduced into the upstream primer, and an Xho I restriction site is introduced into the downstream primer.

[0075] (2) Gene amplification, cloning and recombinant plasmid screening

[0076] The liver cDNA was used as a template, and the above primers were used for PCR reaction. The amplification conditions were: 94°C, 3min pre-denaturation; 94°C, 30s, 53°C, 40s, 72°C, 50s, 35 cycles; 72°C, 10min extension.

[0077] 1% agarose gel electrophoresi...

Embodiment 3

[0084] Example 3: Procambarus clarkii PHB1 recombinant protein has antiviral function

[0085] (1) Quantitative analysis of WSSV copy number by real-time quantitative PCR

[0086] Procambarus clarkii were randomly divided into 6 groups (30 in each group), in which the experimental group was injected with PBH1 co-incubated with white spot syndrome virus (WSSV), and 5 control groups were set up: respectively injected with ① CDK10 co-incubated with WSSV ( Protein recombinantly expressed in the same expression system as PHB1), ②bovine serum albumin (BSA)+WSSV, ③PBS+WSSV, ④inject PHB1 alone, ⑤inject PBS alone. Then the gill tissues of 3 Procambarus clarkii were randomly collected for genomic DNA extraction every day for 7 consecutive days. The gill tissue genome was extracted using the Genome Extraction Kit (MiniBEST Universal Genomic DNA Extraction Kit) from Bao Bioengineering Company (Dalian), and then diluted 10 times as a template for real-time quantitative PCR. Standard curv...

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PUM

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Abstract

The invention discloses procambarus clarkia inhibin (prohibin 1) gene with a nucleotide sequence as shown in SEQ ID NO.1. The invention further provides inhibin coded by the crawfish inhibin 1 gene with the nucleotide sequence as shown in SEW ID NO.2. The invention further provides a use of the gene sequence and a use of the inhibin. The procambarus clarkia inhibin 1 disclosed by the invention can be used for preparing a prokaryotic expression vector, so that escherichia coli is converted and recombinant protein with antiviral activity is expressed. The recombined PHB (Polyhydroxybutyrate) obtained by utilizing the procambarus clarkia inhibin 1 gene can be used for antiviral feed additive, animal and plant gene transformation and medicinal development.

Description

technical field [0001] The invention relates to a Procambarus clarkii inhibitin 1 (prohibitin1) gene and its coded prohibitin 1 protein and its application, in particular to its application in preparing a recombinant protein with antiviral activity, which belongs to the technical field of genetic engineering. Background technique [0002] According to existing studies, there is no adaptive immunity in invertebrates, but invertebrates have a relatively complete immune defense response, mainly including cellular immunity and humoral immunity. Invertebrates rely on these innate immune defense responses to clear or eliminate pathogens. In recent years, prohibitin (PHB) has been extensively studied as a tumor target protein. At the same time, the study also found that PHB also played an important role in the immune response. PHB protein can bind to a variety of viruses. For example, it is reported that PHB can be used as a receptor for dengue fever virus, and PHB can also bind ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/70
Inventor 王金星兰江风赵小凡
Owner SHANDONG UNIV
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