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Exopalaemon carinicauda livetin binding protein gene and application thereof

A yolk protein and binding protein technology, which is applied to the yolk protein binding protein gene of white shrimp and its application field

Active Publication Date: 2020-05-01
HEBEI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In decapod crustaceans, there is no report on the introduction of foreign proteins into fertilized eggs through a mechanism similar to the above

Method used

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  • Exopalaemon carinicauda livetin binding protein gene and application thereof
  • Exopalaemon carinicauda livetin binding protein gene and application thereof
  • Exopalaemon carinicauda livetin binding protein gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] The construction of embodiment 1 recombinant expression vector

[0028] a. Gene cloning:

[0029] A vitellin-binding protein gene EcVBP (sequence shown in SEQ ID NO.1) was screened from the carapace cDNA library of the white shrimp (purchased in Shazikou Market, Qingdao), and its encoded amino acid sequence is shown in SEQ ID NO. As shown in 2, through the online domain prediction tool (SMART:Main page, http: / / smart.embl-heidelberg.de / ) to predict the domain of its amino acid sequence, it is found that there are three Typical structural domains: EcVBP-PC1, EcVBP-PC2, EcVBP-PC3, and the gene sequences corresponding to the three are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively.

[0030] Use the TAKARA (Product No.: 9108) RNAiso Plus kit to extract the TotalRNA of the white shrimp according to the conventional procedures, and use Tiangen Biochemical Technology Co., Ltd. (hereinafter referred to as Tiangen) FastQuant RT Kit (Product No.: KR106-02) accor...

Embodiment 2

[0076] Example 2 Recombinant Vector Induced Expression and Product Purification

[0077] a. Transform the recombinant vectors pCT7-CHISP6H-EcVBP-PC1-EGFP, pCT7-CHISP6H-EcVBP-PC2-EGFP and pCT7-CHISP6H-EcVBP-PC3-EGFP respectively (the transformation step is carried out according to the conventional operation of the prior art) to express the host bacteria BL21(DE3), cultured in LB medium with ampicillin at a final concentration of 100 μg / mL to an OD600 of about 0.6 (37°C, 250 r / min), and added the inducer isopropyl at a final concentration of 1 mmol / L Base-β-D-thiogalactoside (IPTG) was induced to express for 6h.

[0078] b. After centrifuging the supernatant of the fermentation broth (8000rpm, 10min), add PBS and filter with a 0.45 μm filter membrane, and use nickel ion chelation column chromatography (experimental steps are carried out according to the conventional operation of the prior art) to separate and purify the recombinant protein, and the purified Proteins with a gree...

Embodiment 3

[0079] Example 3 Introduction of foreign protein into fertilized eggs

[0080] EcVBP-PC2 and EGFP recombinant proteins were injected into the body fluid of gonad-mature female P. sinensis using an alcohol-sterilized micro-injector, and allowed to mate and lay eggs under laboratory conditions. On the next day, after the female shrimps conceived eggs, the green fluorescent signal ( figure 2 shown), while no green fluorescent signal was observed in normal fertilized eggs ( image 3 shown). This example confirms the feasibility of VBP as a "molecular truck" to carry exogenous proteins into fertilized eggs, and provides a reference for the introduction of other exogenous proteins into fertilized eggs of crustaceans. foundation.

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Abstract

The invention provides an exopalaemon carinicauda livetin binding protein gene and application thereof. The sequence of the gene is shown in SEQ ID NO.1 in the description. The amino acid sequence encoded by the gene is shown in SEQ ID NO.2 in the description. An exopalaemon carinicauda livetin binding protein comprises three structure domains, namely EcVBP-PC1, EcVBP-PC2 and EcVBP-P3 respectively. The invention discloses application of the gene EcVBP in mediating a foreign protein into a zygote of a shelled animal through the livetin binding protein. By adopting the gene provided by the invention, the foreign protein is successfully mediated into a zygote of neocaridina denticulate sinensis, so that feasibility that VBP as a "molecule truck" carries the foreign protein into the zygote ofthe shelled animal is verified, and a basis is made for gene editing and molecular designing breeding of the shelled animal later.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a yolk protein binding protein gene of white shrimp and its application. Background technique [0002] In my country, decapod crustaceans such as shrimps and crabs are important aquaculture economic species. With the increasing market demand for high-quality aquatic products, shrimps and crabs are playing an increasingly important role in aquaculture. As a key step in aquaculture, the production and cultivation of improved species is an important means to fundamentally improve the quality of aquatic products such as shrimp and crab. With the development of disciplines such as molecular biology and cell biology, the advantages of new breeding methods such as transgenic breeding are gradually emerging. [0003] Gene knockout and gene knockin technology is an important means of gene research, and the CRISPR / Cas system emerging in early 2013 is an effective tool to reali...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C07K14/435A01K67/033
CPCC07K14/43509C12N15/70A01K67/0338A01K2207/10A01K2227/70A01K2267/02
Inventor 张继泉邢珂凡刘玉洁孙玉英
Owner HEBEI UNIVERSITY
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