Recombinant protein preparation with anti-aging activity as well as preparation method and application thereof

Abstract

The invention discloses a recombinant protein preparation with anti-aging activity and a preparation method and application thereof, and relates to the technical field of protein preparations, the recombinant protein preparation comprises a recombinant royal jelly major protein rMRJP3, the amino acid sequence of the recombinant royal jelly major protein rMRJP3 is shown as SEQ ID NO.3, and the nucleotide sequence of the recombinant royal jelly major protein rMRJP3 is shown as SEQ ID NO.4. The preparation method comprises the following steps: S1, constructing a prokaryotic expression vector of the recombinant royal jelly major protein rMRJP3; s2, carrying out prokaryotic induced expression on the recombinant royal jelly main protein rMRJP3; and S3, purifying the protein of the recombinant royal jelly main protein rMRJP3 and obtaining the preparation. The recombinant royal jelly main protein rMRJP3 protein preparation can play an anti-aging role, and the life of drosophila melanogaster is remarkably prolonged by 38.36%.

Description

Technical Field

[0001] The present invention relates to the technical field of protein preparations, and in particular to a recombinant protein preparation with anti-aging activity and a preparation method and application thereof. Background Art

[0002] Royal jelly, also known as royal jelly, is a yellowish milky substance secreted by the maxillary glands and subpharyngeal glands of worker bees in a honey bee colony. Its pH value is between 3.4 and 4.5, and it is acidic, sour and astringent. It has a complex chemical composition and significant biological activity. Its main chemical components include: total protein content of about 9% to 18%, total sugar content of about 7% to 18%, lipid content of about 3% to 8%, mineral content of about 0.7% to 1.5%, royal jelly acid content of about 1.4%, ash content of about 0.8% to 3.0%, and water content of about 60% to 70%.

[0003] Major Royal Jelly Proteins (MRJPs) account for 90% of the total protein content of royal jelly. Major royal jelly proteins (MRJPs) are composed of proteins encoded by multiple different genes. Among them, MRJP1, MRJP2 and MRJP3 have the highest content, accounting for 5.89%, 1.41% and 1.66% of fresh royal jelly respectively.

[0004] Existing studies have shown that the main royal jelly proteins (MRJPs) have multiple biological functions. For example, CN114698587A discloses a method for establishing a model and use of MRJPs for regulating mouse immunity and intestinal microorganisms, and clarifies that MRJPs have the function of enhancing mouse immunity and promoting the proliferation of intestinal probiotics. CN104540516A discloses an anti-inflammatory protein and peptide, as well as their preparation methods and uses. The main royal jelly protein shows enhanced anti-inflammatory effects after chemical modification with methylglyoxal. CN115991760A discloses a C-terminal recombinant protein of Western honey bees MRJP3, a preparation method and its application, and successfully constructs a 293F cell expression system of Western honey bees MRJP3 C-terminal recombinant protein. The C-terminal recombinant protein of Western honey bees MRJP3 expressed by the system can significantly promote wound healing. The present invention aims to explore more biological functions of the main royal jelly proteins (MRJPs) and develop a prokaryotic expression recombinant royal jelly main protein. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the present invention provides a recombinant protein preparation with anti-aging activity and a preparation method and application thereof. The prepared recombinant royal jelly main protein rMRJP3 protein preparation has anti-aging activity.

[0006] In order to achieve the purpose of the present invention, the following scheme is proposed: A recombinant protein preparation with anti-aging activity comprises recombinant royal jelly major protein rMRJP3, whose amino acid sequence is shown in SEQ ID NO.3 and nucleotide sequence is shown in SEQ ID NO.4.

[0007] The method for preparing the recombinant protein preparation having anti-aging activity comprises the following steps: S1: Construction of prokaryotic expression vector of recombinant royal jelly major protein rMRJP3; S2: Prokaryotic induction expression of recombinant royal jelly major protein rMRJP3; S3: Purification of recombinant royal jelly main protein rMRJP3 and preparation of the product.

[0008] The recombinant protein preparation with anti-aging activity is used in the preparation of anti-aging preparations.

[0009] The beneficial effects of the present invention are: among the three royal jelly proteins with the highest content, the royal jelly protein MRJP3 has significantly higher anti-aging activity on fruit flies than MRJP1 and MRJP2, so the royal jelly protein MRJP3 can be used to develop and prepare anti-aging preparations; the prepared recombinant royal jelly protein rMRJP3 protein preparation can play an anti-aging effect and significantly increase the lifespan of fruit flies by 38.36%, providing new ideas and methods for the development of new bee products and new anti-aging preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A, B, and C are mrjp1 , mrjp2 , mrjp3 Gene PCR amplification results, where the M column is the nucleic acid molecular weight standard; Figure 2 A, B, and C are the identification diagrams of the prokaryotic expression recombinant vectors pET30-rMRJP1, pET30-rMRJP2, and pET30-rMRJP3, respectively, where M is the nucleic acid molecular weight standard; Figure 3 A, B, and C are the prokaryotic induced expression diagrams of the recombinant protein preparations rMRJP1, rMRJP2, and rMRJP3, respectively, where M is the protein molecular weight standard; Figure 4 A in the middle is the identification diagram of the prokaryotic induction expression form of the recombinant protein preparation rMRJP1; column M in B is the protein molecular weight standard, columns 1 and 2 are the identification diagrams of the prokaryotic induction expression form of the recombinant protein preparation rMRJP2, and columns 3 and 4 are the identification diagrams of the prokaryotic induction expression form of the recombinant protein preparation rMRJP3; Figure 5A, B, and C are the purity detection diagrams of the recombinant protein preparations rMRJP1, rMRJP2, and rMRJP3 by SDS-PAGE electrophoresis, respectively, where M is the protein molecular weight standard; Figure 6 This is a graph showing the effects of different recombinant protein preparations on the survival time of Drosophila; Figure 7 A diagram showing the effects of different recombinant protein preparations on Drosophila lifespan. DETAILED DESCRIPTION

[0011] As one aspect, this embodiment provides a royal jelly major protein MRJP3, whose amino acid sequence is shown in SEQ ID NO.1, and whose nucleotide sequence is shown in SEQ ID NO.2.

[0012] On the other hand, this embodiment provides a recombinant protein preparation with anti-aging activity, including recombinant royal jelly major protein rMRJP3, whose amino acid sequence is shown in SEQ ID NO.3 and nucleotide sequence is shown in SEQ ID NO.4.

[0013] The method for preparing a recombinant protein preparation comprises the following steps: S1. Construction of prokaryotic expression vector of recombinant royal jelly major protein rMRJP3 Total RNA from the head of honey bees was extracted and reverse transcribed into cDNA. Using this as a template, amplification primers were designed according to the full-length gene sequence of the tested royal jelly main protein MRJP3. The specific primers for the MRJP3 gene are as follows: MRJP3-F: 5'-cgGAATTCgtgaatcatcaaagaaaatctgcaaa-3', MRJP3-R: 5'-atttGCGGCCGCatgtaattttgaagaatgatgaacttg-3'; PCR amplification was then performed, and a single band of 1640 bp appeared at the expected position (such as Figure 1 Then, a large-scale PCR amplification was performed to obtain a large amount of mrjp3 Gene fragments were cut and recovered by gel excision. Ecor Ⅰ and Not Ⅰ double enzyme digestion, and finally connected with the pET30 prokaryotic expression vector to construct the prokaryotic expression recombinant vector pET30-MRJP3.

[0014] The same method was used to construct prokaryotic expression recombinant vectors pET30-MRJP1 and pET30-MRJP2.

[0015] MRJP1 gene-specific primers are as follows: MRJP1-F:5'-cgGAATTCattcttcgaggagagtctttaaacaa-3' MRJP1-R:5'-atttGCGGCCGCcaaatggattgaaattttgaaaggtg-3' PCR amplification diagram Figure 1 As shown in A.

[0016] MRJP2 gene-specific primers are as follows: MRJP2-F:5'-cgGAATTCattgttcgagaaaattctccaagaaa-3' MRJP2-R:5'-atttGCGGCCGCtacgcaatgagtatttcttattaattc-3' PCR amplification diagram Figure 1 As shown in B.

[0017] S2. Prokaryotic expression of recombinant royal jelly major protein rMRJP3 The recombinant plasmid was transferred into the competent cells of E. coli Top10, and after heat shock, it was spread on the plate containing the corresponding antibiotic for culture; 10 to 20 single colonies were randomly selected in 600 μL LB liquid culture medium (kana resistance), and cultured at 37°C and 180 r / min for 8h to 12h until the bacterial solution became turbid, and the turbid bacterial solution was subjected to bacterial solution PCR detection; after verification by colony PCR, the positive clone plasmid was obtained by plasmid extraction, and then the obtained positive clone plasmid was transferred into E. coli BL21 after sequencing verification, and the positive expression strain was obtained after plate screening and PCR detection after bacterial culture (the results are shown in the figure). Figure 2 10 μL of the two bacterial cultures that were tested correctly were taken into two glass tubes containing 5 mL of kana-resistant LB liquid medium, and two glass test tubes were inoculated in each tube. Subsequently, the positive expression strains were induced with a final concentration of 0.1 mM IPTG at 37°C for 8 h (the control group was not added with IPTG), and the induced expression was analyzed by SDS-PAGE electrophoresis (as shown in Figure 3 As shown in C), the differential band that appeared near the molecular weight of 77KDd was consistent with the predicted molecular weight of the target protein, indicating that the prokaryotic induction expression of the recombinant royal jelly main protein rMRJP3 was successful. At 37°C, the positive expression strain was expanded to the logarithmic phase, and then induced to express with a final concentration of 0.1 mM IPTG. The bacteria were collected and suspended in 200μl PBS. The bacteria were broken with an ultrasonic disruptor and the supernatant and precipitate were collected by centrifugation, and then the precipitate was suspended with an equal amount of PBS. SDS-PAGE analysis was performed on the sample to be tested, and the recombinant royal jelly main protein rMRJP3 (such as Figure 4 The recombinant protein was mainly expressed in the form of inclusion bodies.

[0018] The same method was used to induce the expression of recombinant royal jelly proteins rMRJP1 and rMRJP2 (such as Figure 2-4 (as shown in A and B).

[0019] S3. Purification and preparation of recombinant royal jelly main protein rMRJP3 The prokaryotic expression Escherichia coli was dissolved by adding 2 volumes of Binding buffer and ultrasonically disrupted, and the crude protein in the supernatant was collected by centrifugation; 1 ml of Ni-NTA was taken, and the column was first washed and balanced with a Banding buffer containing 8 M urea for 10 min at a flow rate of 1 ml / min; the crude protein was incubated with the balanced column filler for 1 h; the incubated product was loaded onto the column and the effluent was collected; the column was washed with a Binding buffer containing 8 M urea for 15 min; the elution was eluted with an Elution buffer containing 8 M urea, and the effluent was collected, which was the protein preparation of the recombinant royal jelly main protein rMRJP3. The purity of the preparation was further detected by SDS-PAGE electrophoresis (such as Figure 5 The protein concentration of the preparation was determined to be 1 mg / ml.

[0020] The same method was used to purify and prepare the protein preparations of recombinant royal jelly main proteins rMRJP1 and rMRJP2 (the results are shown in Figure 5 (as shown in A and B).

[0021] S4. Anti-aging activity test of the protein preparation of recombinant royal jelly main protein rMRJP3 on Drosophila The purified rMRJP3 was added to Drosophila culture medium (each 700 ml culture medium was composed of 82 g corn flour, 62 g sucrose, 10 g agar powder, 7 g yeast powder, 1 ml of 1 mg / ml propionic acid solution was added, and the volume was fixed to 700 ml with double distilled water) at a final concentration of 0.05 mg / ml for Drosophila lifespan test (the culture medium of the blank control group was not added with protein, and the culture medium of the intervention group was added with 0.05 mg / ml casein, rMRJP1, rMRJP2, and rMRJP1+2+3, respectively. rMRJP1+2+3 is a mixed protein preparation prepared according to the corresponding ratio of 4.2:1:1.2 of MRJP1, MRJP2, and MRJP3 in natural royal jelly). 300 newly emerged Drosophila were divided into 6 culture bottles, with 50 flies in each bottle. The culture medium was changed every four days, and the number of dead and surviving Drosophila was recorded every day (such as Figure 6 , Figure 7, as shown in Table 1). The results showed that the survival rate of fruit flies in the rMRJP3 intervention group was significantly higher than that in the casein, rMRJP1, rMRJP2, and rMRJP1+rMRJP2+rMRJP3 intervention groups, and the average lifespan was extended by 38.36% compared with the blank control group. The results showed significant differences, indicating that the rMRJP3 protein preparation has significant anti-aging activity on fruit flies.

[0022] Table 1 Effects of different recombinant protein preparations on the lifespan of Drosophila In summary, the protein preparation of rMRJP3 based on prokaryotic expression of recombinant royal jelly major protein and its preparation technology developed by the present invention has a protein concentration of 1 mg / ml and can prolong the lifespan of fruit flies by 38.36%.

[0023] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be the only ones or to limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention for commercial purposes or profit-making activities without departing from the scope of the present invention are within the scope of protection of the present invention.

Claims

1. A recombinant protein preparation with anti-aging activity, characterized in that: It comprises recombinant royal jelly major protein rMRJP3, the amino acid sequence of which is shown in SEQ ID NO.3, and the nucleotide sequence of which is shown in SEQ ID NO.

4.

2. The method for preparing a recombinant protein preparation having anti-aging activity according to claim 1, characterized in that: The following steps are involved: S1: Construction of prokaryotic expression vector; S2: Prokaryotic induction expression of recombinant royal jelly major protein rMRJP3; S3: Purification of recombinant royal jelly main protein rMRJP3 and preparation of the product.

3. The use of the recombinant protein preparation with anti-aging activity as claimed in claim 1, characterized in that: Used in the preparation of anti-aging preparations.

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