Cloning Of Royal Jelly MRJP1 Gene From Apis Cerana Cerana And Its Expression In Escherichia Coli And Pichia Pastoris And Its Nutritional Function Study

Royal jelly(RJ) is a glandular secretion produced by nurse honeybees to nourish honeybee larvae during their first three days of life and as the sole food for the queen during its life span.RJ has lots of nutritional and biological activities which are closely related with its complex chemical components. Protein is a principal component of RJ and makes up about half of its dry matter.It was divided into two major categories,water-soluble and water-insoluble.Water-soluble protein called major royal jelly proteins(MRJPs) makes up 82—90%of total protein.MRJP1 is the most abundant functional fraction, occupied 48%of MRJPs,it is a 56-57kD protein regarded as the important quality indicators and functional component just like RJ acid.The main results in this study are shown as following:Firstly,three clones of AccMRJP1 were screened out from the sequenced brain cDNA library of Chinese honeybee according to the Expression Sequence Tag(EST) of AccMRJP1.Through identifying with polymerase chain reaction(PCR) and sequencing,a AccMRJP1 clone containing an open reading frame(ORF) of 1302 nucleotides encoding a protein of 433 amino acids was determined. The AccMRJP1 had 99.8%similarity with the previously reported AccMRJP1 sequence(AY279539) in amino acid sequences.Secondly,the AccMRJP1 was sub-cloned into the prokaryotic expression vector pGEX-4T-2 for fusion expression in E.coli BL21.Analysis result of the SDS-PAGE showed that the expression product contained a specific band of protein about 76 kDa in size and accumulated up to about 17.7% of the total bacterial proteins.The fusion protein was cross reactive with GST antibody and purified through affinity chromatography,which confirmed the successful expression of GST-AccMRJP1. And the AccMRJP1 was also sub-cloned into the Eukaryotic expression vector pPIC 9K for fusion expression in Pichia pastoris GS115.Analysis result of the SDS-PAGE showed that the expression product contained a specific band of protein about 58 kDa in size.The fusion protein was cross reactive with His antibody and AccMRJP1 polyclonal antibody,which confirmed the successful expression of His-AccMRJP1.Besides,we completed the preparation of antibody AccMRJP1 which provide an effective tool to detect the other proteins of MRJPs and their expression product. At last,we purified AccMRJP1 with ammonium sulfate and studied its effects on the growth of Tn-5B1-4 cell.The results show that AccMRJP1 has a certain nutritional function and could stimulate cell growth.It is possible for AccMRJP1 to replace FBS in cell cultures.The AccMRJP1 was sub-cloned into the prokaryotic expression vector and the Eukaryotic expression vector successfully which provided a technical base for the utilization of AccMRJP1 with biological engineering.The antibody AccMRJP1 provided an effective tool to detect the other proteins of MRJPs and their expression product.The study also show that AccMRJP1 could stimulate cell growth.which is possiblly used in cell cultures instead of FBS.