Method for preparing MRJP1 eucaryon expression products of China honey bee for promoting growth of cells

A Chinese honeybee, expression product technology, applied in biochemical equipment and methods, botanical equipment and methods, microorganisms, etc., can solve the differences in biological characteristics of natural products, lack of phosphorylation and protein cleavage processing modification process, MRJP1 amount and Solubility is not very ideal and other problems, to achieve the effect of promoting cell growth

Inactive Publication Date: 2008-10-22
ZHEJIANG UNIV
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  • Abstract
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Problems solved by technology

Generally expressed in Escherichia coli, but due to the lack of proper glycosylation, phosphorylation and protein cleavage processing and modification processes, the biological characteristics of the expressed product

Method used

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  • Method for preparing MRJP1 eucaryon expression products of China honey bee for promoting growth of cells
  • Method for preparing MRJP1 eucaryon expression products of China honey bee for promoting growth of cells
  • Method for preparing MRJP1 eucaryon expression products of China honey bee for promoting growth of cells

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Experimental program
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Effect test

Embodiment 1

[0037] The preparation method of the eukaryotic expression product of Apis cerana MRJP1 that promotes cell growth is characterized in that the steps are as follows:

[0038] 1) Cloning of MRJP1 gene

[0039] a) Using the MRJP1 cDNA from the head of the 8-day-old worker bee of Apis cerana as a template, use Primer Primer 5.0 software to design the upstream primer F-Prime sequence as 5' CCATGG ACATGACAAGGTGGTTGTT-3', the downstream primer R-Prime sequence is 3'-TAAAGTTATGTAGACATT CGCC GGCG -5', respectively introduce restriction sites Nco I and Not I into the upstream primer F-Prime sequence and the downstream primer R-Prime sequence (see the underlined part);

[0040] b) The PCR reaction system is 50 μl in total, and the composition is as follows: 5 μl 10×PCR Buffer, 6 μl 25mM MgCl 2 , 1μl 10mM dNTP Mix, 2μl 10μM F-Prime, 2μl 10μM R-Prime, 5μl Apis cerana MRJP1cDNA template, 0.75μl 5U / μl Taq DNA Polymerase, add ddH 2 O to a final volume of 50 μl;

[0041] The PCR reactio...

Embodiment 2

[0051] The eukaryotic expression product of Apis cerana MRJP1 has the function of promoting cell growth, the test is as follows:

[0052] The silkworm cell BmN was cultured in HyQ SFX-INSECT insect cell medium containing 10% FBS at a constant temperature of 27°C to a density of 1.2×10 5 cells / ml, aliquoted to 96-well plates for experiments. A total of 6 experimental groups were designed, 1 blank control group, and each group had three repetitions. The number of cells, test factors and their dosage in each group of experiments were 1×10 4 cells / well, Apis cerana MRJP1 (referred to as rAccMRJP1), 0.1mg / ml; 0.8×10 4 cells / well, rAccMRJP1, 0.5mg / ml; 1×10 4 cells / well, rAccMRJP1, 0.5mg / ml; 1.2×10 4 cells / well, rAccMRJP1, 0.5mg / ml; 1×10 4 cells / well, rAccMRJP1, 1mg / ml; 1×10 4cells / hole, royal jelly water soluble (RJ), 0.5mg / ml; 1×10 4 cells / hole, ddH 2 O. Make up to 100μl on the basis of the original volume, and finally use ddH for each group 2 Make up to 100μl with O, incu...

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Abstract

The invention discloses a method for preparing an Apis cerana MRJP1 eucaryon expression product for promoting cell growth. The method is as follows: firstly, construction of a Bac-to-Bac/BmNPV rhabdovirus expression system is utilized for obtaining recombinant Bacmid-MRJP1 which is then transfected to silkworm cells; secondly, after recombinant viruses are obtained, silkworm larvas are injected; thirdly, after the silkworm larvas are transfected for 96 hours, haemolymph is collected; fourthly, after purification of an affinity column, the high-purity recombinant MRJP1 is obtained. As shown by SDS-PAGE and Western-blotting analysis, the molecular weight of the expression product is approximately 60kDa; molecules are mainly in the state of dissolution; the expression amount is obviously higher than prokaryotic expression. The method establishes certain foundation for industrialized production of the MRJP1 and is hopeful to be applied in the fields such as biomedicine and so on for serving the human health.

Description

technical field [0001] The invention relates to a preparation method of a Chinese honeybee MRJP1 eukaryotic expression product that promotes cell growth. Background technique [0002] Royal Jelly (RJ, Royal Jelly) is a pulpy substance secreted by the hypopharyngeal and maxillary glands of feeding bees for the consumption of queen bees and bee larvae within 3 days. The water content in royal jelly is generally 64.5%-68.5%, protein 12%-14%, carbohydrate 8.5%-16%, lipid 6%, ash 0.4%-2%, and other substances 2.84%-3%. Wherein royal jelly protein is made up of water-soluble protein and water-insoluble protein, and water-soluble protein accounts for 46%~89% of total protein content, is the main part of royal jelly protein, is called main royal jelly protein (MRJPs, Major Royal Jelly Proteins) (Chen Shenglu et al. , 2001; Townsend et al., 1940). The biological activity of royal jelly is closely related to its main protein. [0003] In recent years, the research on apalbumin, esp...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/866C07K14/435C12N5/06
Inventor 苏松坤陶挺缪云根陈盛禄
Owner ZHEJIANG UNIV
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