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A kind of recombinant river crab esflol protein with binding activity to chh and its application

A technology that combines activity and protein, applied in the biological field, can solve problems such as major disputes, and achieve the effect of realizing profits, maximizing and shortening the breeding cycle

Inactive Publication Date: 2018-11-06
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although flotilin is highly conserved in interspecies evolution, its exact function is still controversial and further evidence is needed to support many inferences

Method used

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  • A kind of recombinant river crab esflol protein with binding activity to chh and its application
  • A kind of recombinant river crab esflol protein with binding activity to chh and its application
  • A kind of recombinant river crab esflol protein with binding activity to chh and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Prokaryotic Expression and Purification of Esflol in Eriocheir sinensis

[0039] The prokaryotic expression vector is PET-30 vector, and the expression strain is Rosetta strain. Etaq enzyme, DNA markerDL2000, pMD19-T vector, Nde I, EcorⅠ, DNA Ligation kit were all purchased from TaKaRa Company; agarose, SanPrep column type DNA mini-extraction kit, SanPrep column type plasmid DNA mini-extraction kit , LB solid medium, LB liquid medium, isopropanol, isopropyl-B-D-thiogalactoside (IPTG), glycine, EDTA, SDS, TEMED, Acrylamide (40%), ammonium persulfate, Coomassie Reagents such as blue R-250, bromophenol blue, and Tris were purchased from Shanghai Sangon Bioengineering Co., Ltd.; protein markers were purchased from Fermentas; other reagents were of domestic analytical grade.

[0040] 1.1 Construction of expression vector

[0041] (1) Design PCR primers

[0042] F1: 5'-AATTCCATATGATTCCCATTTTCATCA-3';

[0043] R1: 5'-AATTCTCAGTGGTGGTGGTGGTGGTGTGCTACACGGAGGTT-3',

[0044] ...

Embodiment 2

[0075] Pull-Down experiment of Esflol and CHH

[0076] (1) The prokaryotic expression system constructed by using the constructed Esflol and CHH was expressed, purified, and refolded to obtain two target proteins dissolved in TBS solution, which were stored in a -80°C ultra-low temperature refrigerator for later use.

[0077] (2) Take out the protein sample stored in the ultra-low temperature refrigerator, melt it at room temperature, detect the protein concentration with Nanodrop, and place it on ice for later use;

[0078] (3) Assemble the three sets of prepacked columns and recovery tubes, and mark them as T1, C1, and C2 respectively;

[0079] (4) Prepare 30ml wash solution: mix TBS solution and Pierce Lysis Buffer according to the ratio of 1:1-1:3, and add 3-6M imidazole Stock Solution, so that the final concentration of imidazole in the wash solution is 10mM;

[0080] (5) Mix the HisPur Cobalt Resin filler with a vortexer, and pour 50 μL of the fully mixed filler into ea...

Embodiment 3

[0104] result

[0105] 1. Prokaryotic expression of Esflol in Chinese mitten crab

[0106] 1.1 Construction of Esflol prokaryotic expression vector

[0107] In this experiment, the PET30-Esflol expression vector was successfully constructed, and the recombinant Esflol protein was successfully obtained through the prokaryotic expression of the Rosetta strain. In the process of constructing prokaryotic expression system, the selection of expression strain is very important. Esflol has a relatively large molecular weight and is a membrane protein. It is difficult to express and obtain it through prokaryotic. The codon preference of eukaryotic cells is different from that of prokaryotic systems. Therefore, when using prokaryotic systems to express eukaryotic genes, some codons in eukaryotic genes may be rare codons for prokaryotic cells, resulting in expression efficiency and expression levels are very low. Esflol protein has such a rare codon. Therefore, the Rosetta strain w...

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Abstract

The invention discloses a recombinant river crab Esflol protein with binding activity on CHH and application. The protein has 426 amino acids which are as shown in SEQ ID NO. 1. A theoretical isoelectric point of the coded protein is 9.150, and the molecular weight is 47.18kD; and the protein has a nucleotide sequence which is shown in the SEQ ID NO. 1, and overall length of an open reading frame (ORF) in a corresponding gene sequence is 1281bp. An experiment result shows that a deduction is further verified by an immunohistochemical result of a river crab hepatopancreas tissue slice. Moreover, gene sequence analysis shows that the Esflol protein contains two possible cAMP or cGMP cyclin-dependent kinases phosphorylation sites, and the result prompts that the Esflol possibly participates in a signal channel mediated by cAMP / cGMP. The result proves that the Esflol is a link in the signal channel which is regulated and controlled by the CHH. The recombinant river crab Esflol protein with the binding activity on the CHH is peculiarly combined to the CHH, and nutritive value and flavor of hepatopancreas of cooked crabs are improved.

Description

[0001] This research is supported by the National Natural Science Foundation of China Youth Fund (31302168); Tianjin Applied Basic and Frontier Technology Research Program (General Project) (14JCYBJC30700); Tianjin Normal University Doctoral Fund (52XB1303). technical field [0002] The invention belongs to the field of biotechnology, in particular to a recombinant river crab Esflol protein with binding activity to CHH and its application. Background technique [0003] Crustacean hyperglycemic hormone (CHH) is a member of the neuropeptide hormone family. This protein is only found in arthropods and plays a key role in the regulation of hemolymph glucose levels, molting and stress. Although guanylate cyclase (GC) on the cell membrane has been recognized as a CHH receptor in the Y organ, the receptor in the hepatopancreas remains unidentified. In this study, we screened and cloned a gene whose expression level increased by 2.8 times after eye-stalk removal by analyzing the tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/11
CPCC07K14/43509
Inventor 李冉孙金生耿旭云张亦陈田金泽朱丽娜
Owner TIANJIN NORMAL UNIVERSITY
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