Gene engineering preparation and identification method for eriocheir sinensis crustacean hyperglycemic hormone

A hyperglycemic hormone and gene technology, which is applied in the field of genetic engineering preparation and identification of Chinese mitten crab hyperglycemic hormones, and can solve problems such as raising blood sugar.

Inactive Publication Date: 2015-05-27
TIANJIN NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, there are only some reports on the research on the hyperglycemia hormone of Chinese mitten crab. Zhou Kaiya et al. cloned the partial sequence of the hyperglycemic hormone gene of Chinese mitten crab; Kang Xianjiang et al. The instrument separated the substance that can increase blood sugar concentration. The in vivo experiment of river crab showed that it was glucagon or contained glucagon. After analyzing some of its biochemical properties, it was determined that it was a thermally stable substance with the effect of increasing blood sugar and the acidic isoelectric point.

Method used

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  • Gene engineering preparation and identification method for eriocheir sinensis crustacean hyperglycemic hormone
  • Gene engineering preparation and identification method for eriocheir sinensis crustacean hyperglycemic hormone
  • Gene engineering preparation and identification method for eriocheir sinensis crustacean hyperglycemic hormone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of Prokaryotic Expression Vector of Crab Hyperglycemia Hormone Gene

[0039] The cloning vector pMD18-T was purchased from TaKaRa, the expression vector pCR?T7 / NT TOPO?TA was purchased from Invitrogen, E. coli TOP-10F' and expression host strain BL21(DE3) plysS competent cells were purchased from Beijing Tiangen Biochemical Technology Co., Ltd. DNA polymerase, T4 DNA ligase, endonuclease BamH , Endonuclease Bst B , DNTP, DNA marker DL2000 were purchased from TaKaRa Company, Plasmid Mini Kit, Agarose gel DNA extraction Kit, LB medium were purchased from Shanghai Bioengineering Co., Ltd.; other reagents were domestic or imported analytical pure products. The primers used in the experiment were Es-CHHFM1 (5'-CATATGCAGGCCTACGACCGC-3') and Es-CHHRM1 (5'-AAGCTTTCAGTGGTGGTGGTGGTGGTGGCCAACCACCCGGA-3'), synthesized by Shanghai Shenggong.

[0040] (1) PCR amplification and product purification:

[0041] According to the cDNA sequence of the mature peptide co...

Embodiment 2

[0061] Example 2: Induced expression of recombinant protein and preliminary analysis of expression products

[0062] Main reagents needed: isopropanol, isopropyl-BD-thiogalactoside (IPTG), glycine, SDS, TEMED, acrylamide, methylene bisacrylamide, ammonium persulfate, Coomassie brilliant blue R-250 , Bromophenol Blue, DTT, Glycerin, Tris and other reagents were purchased from Shanghai Shenggong Biological Engineering Co., Ltd.; protein markers were purchased from Fermentas Company; other reagents were all domestic or imported analytical grades.

[0063] The main equipment needed: protein electrophoresis tank, decolorizing shaker (Beijing Liuyi Instrument), low temperature centrifuge (Ependdorf), etc.

[0064] The bacterial liquid containing the expression vector stored in Example 1 was inoculated into 20 ml of fresh LB liquid medium containing ampicillin (containing 100 μg / mL ampicillin), and cultured at 37° C. until the OD600 reached 0.6-0.8. Take 1ml of the culture medium without ...

Embodiment 3

[0068] Example 3: Separation, purification and renaturation of recombinant protein

[0069] (1) Broken bacteria:

[0070] Ferment the bacteria stored in Example 1 to an OD600 nm of 0.6-0.8, add IPTG with a final concentration of 1 mmol / L to induce culture for 5 h, then take 300 ml of the bacteria liquid and centrifuge at 10,000 rpm for 10 min at 4 ℃ to collect the bacteria . Resuspend the bacteria at the ratio of 1g wet bacteria / l0ml disrupting buffer (1% TritonX-100, 2 mM EDTA), and ultrasonically disrupt under ice bath conditions (working conditions: power 400 W, working 5s, intermittent 5s, total 15min , Repeated 3 times), collect the inclusion bodies by centrifugation at 10,000 r / min for 10 min at 4 ℃ after the crushing is completed.

[0071] (2) Washing of inclusion bodies:

[0072] Take the bacterial pellet collected after sonication and add 30ml of inclusion body washing buffer (300mM KCL, 50mM KH 2 PO4, 5mM Iminazole, 1M Urea, pH 7.4) Wash 3 times, collect the precipitate, ...

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Abstract

The invention discloses a gene engineering preparation and mass spectrometry identification method for eriocheir sinensis crustacean hyperglycemic hormone (Ers-CHH). Energy supplies of various tissues and organs are maintained by CHH through regulating a blood sugar level of a crustacean, and researches on family nerve polypeptide hormones can provide helps for revelation of crustacean metabolism, growth and reproduction regulation mechanisms. In previous studies, an eriocheir sinensis crustacean hyperglycemic hormone gene (Ers-CHH, GenBank registration number: JX485664) is cloned. According to the present invention, a gene engineering method is adopted to successfully obtain recombinant Ers-CHH, wherein a molecular weight is about 9.4 kD, a yield after purification renaturation is 0.4 g / L culture medium, and mass spectrometry identification results verify correctness of the recombinant protein. Due to low CHH content in the crustacean, direct purification enrichment on the CHH is virtually impossible, and a large number of the rare hormone proteins are rapidly obtained by using the gene engineering method in the present invention so as to establish a foundation for subsequent function regulation mechanism research development and application in aquaculture production practice.

Description

Technical field [0001] The invention has obtained the National 973 Planning Project (2012CB114405), the National 863 Plan Project (2012AA092205 and 2012AA10A401), the National Science and Technology Support Program Project (2011 Bad13B04 and 2011 Bad13B07), the Tianjin Natural Science Foundation of China (10JCYBJC09200) and Tianjin Normal University Dr. Fund Project (52LX181881881881881881881881881881881881881881881881881881881881881881881881881881881881881881881818Funding with 52lx19). [0002] The present invention involves the field of biotechnology, specifically a Chinese velvet crab -crab hyperglycemia gene ( ERS-CHH ) Gene engineering preparation and appraisal methods. Background technique [0003] Chinese velvet crab is commonly known as river crab and hairy crabs. It is one of the crustaceans with high economic value in my country. It has a delicious taste and rich nutrition. Class, so economic value is increasing day by day. [0004] The X-Organ-Sinus Gland Complex (XO-S...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/70C12N15/11C12N15/85A01K67/027
Inventor 刘逸尘张亦陈孙金生王艳华孙妍耿绪云
Owner TIANJIN NORMAL UNIVERSITY
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