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

A technology of hyperglycemia hormone, Chinese mitten crab, applied in the direction of DNA/RNA fragment, introduction of foreign genetic material and peptide source using a vector, etc., can solve problems such as raising blood sugar

Inactive Publication Date: 2013-09-04
TIANJIN NORMAL UNIVERSITY
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  • Abstract
  • Description
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AI Technical Summary

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

[0039] Example 1: Construction of the prokaryotic expression vector of the river crab hyperglycemia hormone gene

[0040]Cloning vector pMD18-T was purchased from TaKaRa Company, expression vector pCR?T7 / NT TOPO?TA was purchased from Invitrogen Company, Escherichia 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, and DNA marker DL2000 were purchased from TaKaRa Company; Plasmid Mini Kit, Agarose gel DNA extraction Kit, and LB medium were purchased from Shanghai Bioengineering Co., Ltd.; other reagents were domestic or imported analytically pure products. The primers used in the experiment were Es-CHHFM1 (5’-CATATGCAGGCCTACGACCGC-3’) and Es-CHHRM1 (5’-AAGCTTTCAGTGGTGGTGGTGGTGGTGGCCAACCACCCGGA-3’), which were synthesized by Shanghai Sangong.

[0041] (1) PCR amplification and product purification:

[00...

Embodiment 2

[0064] Example 2: Induced expression of recombinant proteins and preliminary analysis of expression products

[0065] Main reagents required: isopropanol, isopropyl-B-D-thiogalactoside (IPTG), glycine, SDS, TEMED, acrylamide, methylenebisacrylamide, ammonium persulfate, Coomassie brilliant blue R-250 , bromophenol blue, DTT, glycerin, Tris and other reagents were purchased from Shanghai Sangon Bioengineering Co., Ltd.; protein markers were purchased from Fermentas; other reagents were of domestic or imported analytical grade.

[0066] The main instruments required: protein electrophoresis tank, decolorization shaker (Beijing Liuyi Instrument), low temperature centrifuge (Ependdorf), etc.

[0067] Inoculate the bacterial solution containing the expression vector preserved in Example 1 into 20 ml of fresh LB liquid medium containing ampicillin (100 μg / mL ampicillin), and culture at 37°C until the OD600 reaches 0.6-0.8. Take 1ml of the culture solution without IPTG induction a...

Embodiment 3

[0074] Example 3: Separation, purification and renaturation of recombinant protein

[0075] (1) Broken bacteria:

[0076] Ferment the bacteria preserved in Example 1 to OD600nm of 0.6~0.8, add IPTG with a final concentration of 1 mmol / L to induce culture for 5 hours, take 300ml of the bacteria liquid and centrifuge at 10000 rpm for 10 minutes at 4°C to collect the bacteria . Resuspend the bacteria at a ratio of 1g wet bacteria / l0ml disruption buffer (1% TritonX-100, 2 mM EDTA), and ultrasonically disrupt under ice bath conditions (working conditions: power 400 W, working for 5s, intermittent for 5s, a total of 15min , repeated 3 times), after the crushing was completed, centrifuge at 10,000 r / min for 10 min at 4°C to collect inclusion bodies.

[0077] (2) Washing of inclusion bodies:

[0078]Take the bacterial pellet collected after ultrasonic disruption, add 30ml of inclusion body washing buffer (300mM KCL, 50mM KH 2 PO4, 5mM Iminazole, 1M Urea, pH 7.4) washed 3 times, ...

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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

[0001] The invention was supported by National 973 Program (2012CB114405), National 863 Program (2012AA092205 and 2012AA10A401), National Science and Technology Support Program (2011BAD13B04 and 2011BAD13B07), Tianjin Natural Science Foundation Project (10JCYBJC09200) and Tianjin Normal University Doctoral Fund Project (52LX18 and 52LX19). technical field [0002] The invention relates to the field of biotechnology, in particular to a Chinese mitten crab hyperglycemia hormone gene ( Ers-CHH ) genetic engineering preparation and identification method. Background technique [0003] Chinese mitten crab, commonly known as river crab and hairy crab, is one of the crustaceans with high economic value in my country. It is delicious and nutritious, rich in protein, vitamin A, riboflavin, niacin, etc., and its nutritional value exceeds that of ordinary fish. class, so the economic value is increasing day by day. [0004] The eye stalk X organ-sinus gland complex (X-Organ-Sinus Gland...

Claims

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Application Information

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Patent Type & Authority Applications(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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