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Yeast expression method for recombining major protein AccMRJP1 of apis cerana royal jelly and product application

A technology of royal jelly major protein and Apis cerana, which is applied in the field of yeast expression of recombinant royal jelly protein AccMRJP1 of Apis cerana to achieve a wide range of effects

Inactive Publication Date: 2011-04-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, there have been many research reports on the expression of human epidermal growth factor by recombinant genetic engineering methods, but there is no domestic use of yeast as a bioreactor to produce royal jelly MRJP1 through gene recombination, let alone yeast expression, glycosylated Modified, AccMRJP1 of Apis cerana with cell growth factor activity

Method used

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  • Yeast expression method for recombining major protein AccMRJP1 of apis cerana royal jelly and product application
  • Yeast expression method for recombining major protein AccMRJP1 of apis cerana royal jelly and product application
  • Yeast expression method for recombining major protein AccMRJP1 of apis cerana royal jelly and product application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment i

[0044] Cloning of embodiment i AccMRJ1 gene

[0045] Material

[0046] All primers were synthesized by Shanghai Sangon Company:

[0047] Example of upstream primer 19k-ecor-6hisek-F: 5′-A CATCATCATCATCATCAT GCATTCTTCGAGGA-3' (sequence shown in SEQ ID NO.1), in which the underlined sequence encodes 6 histidines (6×His), which is convenient for affinity separation of expression products; the framed sequence is the enzyme cleavage site EcoR I, italics is the thrombin cleavage site, which is convenient for cutting 6 histidines from the expression product; A before the cleavage site is the protective base, and the rest is the coding sequence at the 5' end of the mature peptide of AccMRJP1.

example 1

[0048] Downstream Primer Example 1 MRJP-NOT-R: 5’-CCG TTACAGATGTATTGAAATTTTG-3' (sequence shown in SEQ ID NO.2). The framed sequence is the restriction site NotI, the CCG before the restriction site is the protective base, and the rest are complementary sequences at the 3' end.

[0049] The a-factor primer is 5'-TACTATTGCCAGCATTGCTGC-3' (sequence shown in SEQ ID NO.3), designed according to the a-factor at the 5' end of pPIC9K.

[0050] Example of upstream primer 25'AOX1: 5'-GACTGGTTCCAATTGACAAGC-3' (sequence shown in SEQ ID NO.4), designed according to the 5'-promoter sequence of vector pPIC9K, used for PCR detection of recombinant yeast vector.

[0051] Downstream primer example 2AOX1: 5'-GCAAATGGCATTCTGACATCC-3' (sequence shown in SEQ ID NO.5), designed according to the 3'-promoter sequence of vector pPIC9K, used for PCR detection of recombinant yeast.

[0052]Escherichia coli (Escherichia coli) strains TG1 and DH5α are preserved by our laboratory;

[0053] Pichia pasto...

Embodiment 2

[0062] Example 2 Construction and induced expression of highly expressed yeast recombinants

[0063] Preparation of Competent Cells

[0064] ①Stretch culture of GS115 strain on YPD solid medium (YPD liquid medium formula: peptone 20g, yeast extract 10g, glucose 20g, add deionized water 1000mL. YPD solid medium: YPD liquid medium + 1.5 agar powder) , cultured at 37°C for 1-2 days, picked a single colony of yeast, inoculated into a 50mL Erlenmeyer flask containing 5ml of YPD medium, and cultured overnight at 30°C for 250-300min. ②The next day, inoculate 100-500 μl of the culture into a 2L Erlenmeyer shaker flask containing 500 mL of fresh YPD medium, and culture overnight at 28-30°C, 250-300 rpm, until OD 600 The value reaches 1.3-1.5. ③ Centrifuge the cell culture at 4°C and 1500g for 5 minutes, and resuspend the bacterial cell pellet with 500 mL of ice-cold sterile water; ④ Centrifuge according to step 3, and use 250 mL of ice-cold sterile water to pellet the bacterial cell ...

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Abstract

The invention discloses a yeast expression method for recombining a major protein AccMRJP1 of apis cerana royal jelly and product application. The method comprises the following steps of: screening out a clone containing the full length of an AccMRJP1 gene from an apis cerana brain cDNA library by utilizing a genetic engineering means; obtaining a target gene through PCR (Polymerase Chain Reaction) amplification; optimizing the gene to be linked to an Escherichia coli-yeast shuttle vector pPTC9k; linearizing and introducing to a yeast cell; culturing the yeast cell under the condition of inducing AccMRJP1 expression to finally obtain an expression product; and purifying through affinity chromatograph to obtain a product with the same glycosylation degree and biological activity with natural proteins. The recombined AccMRJP1 obtained in the invention can be used for vitro serum-free culture of passage and primary insects as well as human and animal cells, eliminates the risk of numerous unknown cell factors, such as polluted exogenous viruses and pathogenic factors in blood serum and the interference of antigen, antibody, hormone, and the like, and has wide application in the fields, such as life sciences, medicine, biomedicine, and the like.

Description

technical field [0001] The invention relates to the field of yeast genetic engineering, in particular to a method for expressing recombinant Chinese honeybee (Apis cerana cerana) royal jelly protein AccMRJP1 in yeast and the use of the product. Background technique [0002] Royal jelly is a compound secreted by the exocrine glands in the head of adult worker bees. It is an important component of the food used by bee colonies to feed bee larvae, and is the most important nutrient to promote the differentiation of larvae. Protein is one of the main components of royal jelly, accounting for 50% of the dry weight of royal jelly. Generally, royal jelly protein is composed of water-soluble protein and water-insoluble protein, wherein water-soluble protein accounts for 46%-89% of the total protein content, and is the main part of royal jelly protein, called Major Royal Jelly Proteins (MRJPs). In addition, Schmidt et al speculated that the content of MRJPs in royal jelly protein ma...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/81C07K14/435C12N5/07
Inventor 沈立荣赖超强袁鹏莫寅元张伟光张瑮文
Owner ZHEJIANG UNIV
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