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Chinese bee royal jelly main protein AccMRJP7 gene and encoding protein thereof

A royal jelly major protein, technology of Apis cerana, applied in the preparation of polynucleotides and said protein, cDNA sequence of AccMRJP7 gene of royal jelly major protein of Apis mellifera, homologue of AccMRJP7 protein of Apis mellifera, AccMRJP7 gene of royal jelly protein of Apis mellifera and Its coding protein field can solve problems such as failure to obtain full-length gene sequences

Inactive Publication Date: 2009-05-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, genes with relatively small cDNA content in AccMRJPs, such as AccMRJP4, have not been successfully cloned, and only partial sequences of AccMRJP6 and AccMRJP7 have been obtained (Su et al.2005.Apidologie.36:389-401). Get the full-length gene sequence
For example, they only published the nucleotide sequence of 220 amino acid sequences encoding the AccMRJP7 protein in GenBank (GenBank sequence number GI: AY862496), and according to the research results of the inventors, the complete protein of AccMRJP7 actually has 449 amino acids in full length, namely The protein sequence released by Su Songkun et al lacks 249 amino acids, and this part of the sequence is impossible to realize recombinant expression in scientific research and application

Method used

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  • Chinese bee royal jelly main protein AccMRJP7 gene and encoding protein thereof
  • Chinese bee royal jelly main protein AccMRJP7 gene and encoding protein thereof
  • Chinese bee royal jelly main protein AccMRJP7 gene and encoding protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Cloning and determination of the royal jellybumin AccMRJP7 gene of Apis mellifera

[0043] 1. cDNA library construction

[0044] The worker bees that emerged on the same day were marked with a paint pen, recaptured at 1, 3, 4, 5, 7, 9, 12, 15, 18, 21, 24, 27 and 30 days of age, and stored at -80°C. Take 10 heads from each day-old worker bee sample, add liquid nitrogen to homogenate, add TRIZOL to extract total RNA. The mRNA was purified step by step with the kit, and the first and second strands of cDNA were synthesized. Then use end-filling enzyme to fill in the double-stranded cDNA end, add EcoRI linker and phosphorylate it, and then digest it with Xho I. The digested product is identified by agarose gel electrophoresis, and then MinElute Gel Extraction kit Perform gel recovery.

[0045] Connect the cDNA to the carrier pBluescript II SK(+), transform E.coli DH10B competent bacteria by electroshock reaction method, add SOC medium, recover on a shaker at 3...

Embodiment 2

[0053] Example 2: Homologous comparison

[0054] With the full-length cDNA sequence of the Chinese honeybee royal jelly major protein AccMRJP7 of the present invention and its encoded protein, in the Non-redundant GenBank+EMBL+DDBJ+PDB database and the Non-redundantGenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, use The Blast program performs nucleic acid and protein homology searches. As a result, it was found that it has significant homology with the royal jellybumin AmMRJP7 of Apis mellifera (GenBank serial number GI: 40949636). The amino acid sequence alignment results of AccMRJP7 and AmMRJP7 (see Table 1). The lengths of the amino acid sequences of AccMRJP7 and AmMRJP7 are 449aa and 443aa respectively, and the homology of the two is 84.2%. Some sequences corresponding to the Asn site of AccMRJP7 in AmMRJP7 are replaced by Asp, but the content of Asp in the amino acid sequence of AccMRJP7 (6.4%) is only It is lower than AmMRJP7 (8.1%). The C-terminal diffe...

Embodiment 3

[0064] Example 3, Expression of AccMRRJP7 in Escherichia coli

[0065] In this example, the clone with the complete AccMRJP7 nucleotide sequence in Example 1 was used as a template, and a pair of oligonucleotides designed according to the AccMRJP7 nucleotide sequence were used as primers to amplify the AccMRJP7 gene in the large intestine Recombinant expression in Bacillus.

[0066] The 5' oligonucleotide primer sequences used in the PCR reaction are:

[0067] 5' end primer: 5'-GATCCATGGCCATTGTTCGAAAAAATTC-3' (SEQ ID No.1), this primer contains the restriction endonuclease cut site of BamHI, the translation initiation codon and the partial coding sequence of AccMRJP7;

[0068] The 3' end primer sequence is:

[0069] 3'-end primer 5'-TCGAGCTAATTAATTAAAGAGTTTATTG-3' (SEQ ID No.2) This primer contains a restriction endonuclease restriction site for Xho I, a translation terminator and a partial coding sequence of AccMRJP7.

[0070] The restriction endonuclease cutting site on t...

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Abstract

The invention discloses a main protein AccMRJP7 gene of Chinese royal jelly honey and an encoding protein of the gene, the gene has the DNA sequence shown in SEQ ID No. 5, and the encoding protein of the gene has the amino acid sequence shown in SEQ ID No. 6 and can be used for antibody preparation. The main protein AccMRJP7 gene of Chinese royal jelly honey and the encoding protein of the gene establish certain foundation for MRJP7 bioengineering application, particularly provides a new method for detecting MRJP protein family by using a MRJPs member antibody, which has wide usage in the research and production fields of royal jelly. The main protein AccMRJP7 gene of Chinese royal jelly honey and the encoding protein of the gene provide foundation for further research of MRJP-related protein active molecule mechanism as well as development of MRJPs-related functional food, diagnostic reagents and biological reagents.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a gene of royal jellybumin AccMRJP7 of Apis mellifera and its encoded protein. More specifically, the present invention relates to the cDNA sequence of the AccMRJP7 (Main royal jelly protein) gene of Apis mellifera. The AccMRJP7 encoded by the cDNA sequence is a homologue of the Apismellifera AmcMRJP-7 protein. After translation, the AccMRJP7 The resulting protein is a homologue of the Apis mellifera AmcMRJP7 protein (AmcMRJP7). The present invention also relates to the polypeptide encoded by the nucleotide sequence, the application of these polynucleotides and proteins, and the preparation methods of said polynucleotides and said proteins. Background technique [0002] Royal jelly (RJ) 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 t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435C07K16/18
Inventor 沈立荣张传溪李艳邢丽苹程家安
Owner ZHEJIANG UNIV
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