CN102558333A - Giant panda ribosomal subunit gene RPL23a recombinant protein and application thereof in preparing anti-cancer drugs - Google Patents

Abstract

The invention discloses a giant panda ribosomal protein subunit gene RPL23a recombinant protein and its application in preparing anticancer drugs. The amino acid sequence of the recombinant protein is defined by SEQ ID NO:7. The giant panda ribosomal protein subunit gene RPL23a recombinant protein was used to act on Hep-2 cells, and the growth inhibitory effect of the giant panda ribosomal protein subunit gene RPL23a recombinant protein on Hep-2 cells was observed, aiming to reveal the giant panda ribosomal protein subunit The anti-cancer function of the gene RPL23a recombinant protein provides a scientific basis and resources for the research and development of anti-cancer protein drugs and the research of anti-cancer mechanism.

Description

Giant panda ribosomal protein subunit gene RPL23a recombinant protein and its application in the preparation of anticancer drugs

technical field

The invention relates to a giant panda ribosomal protein subunit gene RPL23a recombinant protein and its application in the preparation of anticancer drugs, belonging to the field of biotechnology.

Background technique

Mammalian ribosomes are composed of 4 RNAs and about 80 different proteins. The names of ribosomal proteins are related to the size of the protein in the ribosome. The small subunit ribosomal proteins are named S1 to S31, and the large subunit The base ribosomal proteins are named L1 to LA4. The RPL23a protein is a component of the 60S ribosomal protein, encoded by the rpl23a gene. At present, the research of rpl23a gene in humans and some animals and plants has been reported, and the cloning and analysis of genes such as ribosomal protein subunits S7, S12, S15, S20, S24, S28 and LSM3 of giant pandas have also been reported (Luo Xiaoyan et al., 2008 ; Du Yujie et al., 2009; Hao Yanze et al., 2009; Hou Yiling et al., 2009; Zhang Tian et al., 2009; HouW R et al., 2009; Zhang et al., 2009), while the giant panda rpl23a gene and its RPL23a protein Not yet reported. Especially the anticancer function of the giant panda ribosomal protein subunit gene RPL23a recombinant protein has not been reported at home and abroad.

The giant panda is a unique and rare species in my country and is a national first-class protected animal. It has extremely high ecological, scientific research, economic, cultural and aesthetic values. Scholars at home and abroad have conducted a lot of research on giant pandas from different angles and fields. At present, the research on the functional genes of giant pandas has become a research hotspot at home and abroad (Wang Xiaojing et al., 2002; Hou Wanru et al., 2007; Hou W R et al., 2007; Hou Y L et al., 2009; Qiao et al., 2009; Xu et al., 2009; Wan et al., 2009; Tao et al., 2010).

Contents of the invention

In the present invention, primers are designed according to the known sequences of four mammal species: human ( Homo sapiens ), bovine (Bos Taurus), wild boar (Sus scrofa), and musculus musculus (Musmusculus), using giant panda skeletal muscle as material, using RT -PCR and PCR techniques respectively amplified, cloned and sequenced the expression sequence of the giant panda rpl23a gene; compared and analyzed its sequence, and constructed a recombinant expression vector containing rpl23acDNA, which was transformed into E.coliBL21 for overexpression; Purify the overexpressed product to obtain the recombinant protein of the giant panda ribosomal protein subunit gene RPL23a; use the recombinant protein of the giant panda ribosomal protein subunit gene RPL23a to act on Hep-2 cells, and observe the recombinant protein of the giant panda ribosomal protein subunit gene RPL23a The inhibitory effect of the protein on the growth of Hep-2 cells aims to reveal the anti-cancer function of the recombinant protein of the giant panda ribosomal protein subunit gene RPL23a, and provide scientific basis and resources for the research and development of anti-cancer protein drugs and the research of anti-cancer mechanism.

Description of drawings

Fig. 1 Effect of recombinant protein of giant panda ribosomal protein subunit gene RPL23a on the growth of Hep-2 cells; body protein subunit gene RPL23a recombinant protein concentration);

Fig. 2 The effect of giant panda ribosomal protein subunit gene RPL23a recombinant protein on the morphology of Hep-2 cells; body protein subunit gene RPL23a recombinant protein concentration).

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments.

1.1 Materials and methods

1.1.1 Materials and reagents

Giant panda skeletal muscle tissue was obtained from a dead giant panda at the China Conservation and Research Center for the Giant Panda in Wolong, Sichuan. To prevent RNA degradation, the tissue was immediately frozen and stored in liquid nitrogen; human laryngeal carcinoma Hep-2 cells (purchased from Biochemical and Institute of Molecular Immunology). Total RNA Extraction Kit Total Tissue/cell RNA Extractiob Kits was purchased from Waton Company; Reverse Transcription System was purchased from Promega Company; Gel Recovery Kit was purchased from OMEGA Company; T Vector Kit (PMD-18T Vector Systems) And restriction endonucleases Sma I, PstI, SeaII, EcoR I and Sal I were purchased from Treasure Bio (Dalian) Co., Ltd.; Taqplus polymerase was purchased from Shanghai Bioengineering Company, and other reagents were domestic analytical grade. The host strain E.coli DH5a was preserved by the Key Laboratory of Wildlife Conservation and Utilization of Sichuan Province. CW0009 Ni-Agarose His-tagged protein purification kit was purchased from Beijing Yilaisino Biotechnology Co., Ltd.; Bradford protein concentration assay kit (Mbchem ^TM M2031) was purchased from Shanghai Meiji Biotechnology Co., Ltd.; RPMI1640 powder was purchased from Gibco (USA ), fetal bovine serum was purchased from Sijiqing Biological Products Company, and double antibodies (penicillin and streptomycin were purchased from Gibco Company).

1.1.2 Method

1.1.2.1 Extraction of total RNA

Take about 600mg of sample tissue, fully grind in liquid nitrogen, and extract total RNA according to the method recommended in the extraction kit manual. The extracted total RNA was dissolved in DEPC-treated water and stored at -70°C for future use.

1.1.2.2 Primer design and RT-PCR

Relevant information about the coding sequence of the RPL23a gene in four mammalian species, human (NM_000984), bovine (NM_001045958), wild boar (AK233511), and Mus musculus (NM_207523), was retrieved from the GenBank database. According to the conservation of the coding sequence, using The upstream and downstream primers (respectively: Pd-RPL23a-F, Pd-RPL23a-R) designed by Primer Premier 5.0 software were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The numbers and sequences of the designed primers are as follows:

Pd-RPL23a-F: 5'-CCTTTTCGACAAGATGGCGC-3' (SEQ ID NO: 1)

Pd-RPL23a-R: 5'-GAATTAGCCA GCTGGACTCA-3' (SEQ ID NO: 2)

Using total muscle RNA as a template and _Oiligo (dT) as a reverse transcription primer, synthesize the first strand of cDNA according to the operation manual of the reverse transcription kit. Oiligo(dT) ₁₅ 0.5μg, AMV reverse transcriptase 15U, RNase inhibitor 10U/μL. Reverse transcription conditions: 42°C for 60 minutes; then use an appropriate amount of the first strand product as a template, and use a 25 μL PCR reaction system for PCR amplification. The concentrations of each component are: MgCl ₂ 1.5Mm, dNTP 0.2mM, Pd-RPL23a-F and Pd - 0.3 μM each of RPL23a-R, 5 U of Taq plus polymerase. Reaction conditions: 94°C for 5min, 94°C for 1min, 52°C for 0.5min, 72°C for 1.5min, 32 cycles; 72°C for 10min. Then the RT-PCR products were detected by agarose gel electrophoresis with a mass fraction of 1%, and stored at -20°C.

1.1.2.3 Cloning and identification of cDNA

The PCR product was recovered and purified, and the plasmid pUC18 fragment was digested and blunt-ended with Sma I restriction endonuclease, and the recovered product was ligated with the digested and blunt-ended plasmid fragment, and ligated overnight at 22°C. Transform competent cells according to conventional methods, and then spread them on LB medium containing ampicillin, IPTG and x-gal for overnight culture, pick 5 white colonies with an inoculation needle, extract plasmids by alkaline lysis, and use double enzyme digestion (Pst I and Sca II) and PCR, these two methods were used to identify the recombinant plasmids. The identified positive recombinant plasmids were sequenced by Beijing Huada Biotechnology Development Co., Ltd.

1.1.2.4 Data processing

Genescan (http://genes.mit.edu/GENSCAN.html) software was used to deduce the amino acid sequence of the cloned gene sequence; ORF finder software (http://www.ncbi.nlm.gov/gorf.htlm ) carries out the ORF search of DNA sequence;

1.1.2.5 Cloning of RPL23a cDNA expression fragment

1.1.2.5.1 Design of primers

The restriction endonuclease EcoRI recognition site (GAATTC) and the Sal I recognition site (GTCGAC) were introduced into the forward primer and reverse primer of RPL23a respectively. The numbers and sequences of the designed upstream and downstream primers are as follows:

RPL23abd-F 5'-CA GAATTC ATGGCGCCGAAG C-3' (EcoRI) (SEQ ID NO: 3)

RPL23a bd-R 5'-CG GTCGACTTAGATGATCCCA -3'(SalI) (SEQ ID NO: 4)

Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

1.1.2.5.2 PCR amplification

Using the recombinant plasmid connected with cDNA as a template, the newly synthesized primers RPL23abd-F and RPL23abd-R containing restriction sites were used for PCR amplification reaction. 30 s at ℃, 45 s at 55 ℃, 1 min at 72 ℃, 35 cycles; 7 min at 72 ℃, after the end of PCR, then use 1.0% agarose gel electrophoresis to detect the PCR product and tap the gel to recover the target DNA fragment.

1.1.2.6 Construction of RPL23a gene fusion expression vector

1.1.2.6.1 Obtaining the fusion expression fragment of RPL23a gene

The reaction system is 40ul: 10ul of double-enzyme-digested target DNA fragment, 4.0ul of 10xK buffer, 1.0ul of EcoR I, 1.0ul of Sal I, and 24ul of ddH2O. Reaction conditions: Enzyme digestion at 37°C overnight; after digestion, 65°C water bath for 10 minutes, ice-water bath for 1-2 minutes; 4°C, 12000g, centrifugation for 1 minute; 1.0% agarose gel electrophoresis to detect the digested products, and recover the digested expression fragments. The recovered product was stored at -20°C for future use.

1.1.2.6.2 Treatment of expression vector pET-28a

1.1.2.6.2.1 Small scale preparation of pET-28a vector

A small amount of pET-28a plasmid was extracted from DH5α by alkaline lysis method. The specific steps of small plasmid extraction are as follows:

1.5ml (750ul×2) bacterial solution, 12000g, centrifuge at 4°C for 2min, remove the supernatant, then centrifuge briefly, remove the supernatant thoroughly; add 133ul of lysate buffer I, blow it out with a pipette tip, and mix well; add 133ul of 2% SDS, 133ul 0.4M NaOH, invert and mix 5 times (now use and mix now); add bufferIII 200ul invert and mix, ice water bath for 5min; take out, 12000g 4 ℃, centrifuge for 9min, transfer the supernatant to a new EP tube; 12000g, Centrifuge at 4°C for 5 minutes, transfer the supernatant to a new EP tube; add 2 times the volume of absolute ethanol (800ul), mix upside down; put it in the refrigerator (-20°C) for about half an hour (the time can be longer or Overnight); take out 12000g, 4°C, centrifuge for 9min, remove supernatant; add 70% C _{2 H 5 OH} 1000ul, 12000g, centrifuge for 3min; add 70% C2H5OH 1000ul, 12000g _, centrifuge for 3min, suck The clean supernatant (can be centrifuged and reabsorbed instantaneously); put it into a clean bench and air-dry it (15min); dissolve it with ddH ₂ O (20-25ul), and store it at -20°C for later use.

1.1.2.6.2.2 Preparation of expression vector pET-28a

The digestion reaction system of the expression vector is 40ul: pET-28a expression vector 10ul, 10xK buffer 4.0ul, EcoR I 1.0ul, Sal I 1.0ul, ddH2O 24ul. Reaction conditions: Enzyme digestion at 37°C overnight, take out 65°C water bath for 10min, ice water bath for 1-2min, 4°C, 12000g, centrifuge for 1min, detect the digested product by 1.0% agarose gel electrophoresis, recover the digested expression fragment, and recover the product Store at -20°C for later use.

1.1.2.7 Ligation and transformation of target gene and expression vector

The ligation reaction system is 20ul: RPL23a digestion fragment 2ul, 10xligase buffer 2ul, enzyme digestion expression vector pET-28a 3ul, T4 ligase 1ul, ddH2O 12ul. Total 20ul. Reaction conditions: keep warm overnight at 16°C, keep warm at 65°C for 10 minutes, and terminate the reaction. Transform competent cells DH5a, spread the transformed cell suspension on LB plates [containing X-gal (40ug/mL), IpTG (25ug/mL) and Amp (100ug/mL)], and culture overnight in a 37°C incubator.

1.1.2.7.1 Screening of recombinants and preparation of recombinant plasmids

Randomly pick 5 single colonies (plates with transformation products) for PCR and enzyme digestion identification to ensure the reliability of the screening; at the same time, in a 5ml/50ml conical bottom centrifuge tube, culture at 37°C for 16h, and alkali lysis The recombinant plasmid was extracted by 0.8% agarose electrophoresis.

1.1.2.7.2 Induced expression of pET-RPL23a recombinant vector in Escherichia coli

1.1.2.7.3 Conversion

Transform the recombinant plasmid into expression strain BL21(DE3), the specific operation is the same as 1.1.2.3.

1.1.2.7.4 Inducible expression vector

Commonly used induction expression methods include temperature induction and drug induction. In this experiment, IPTG (isopropyl-bD-thiogalactopyranoside, isopropyl-bD-thiogalactopyranoside) was used to induce the expression of exogenous genes. The specific operation steps: the bacterial liquid Transfer to 100ml LB medium + Kan (80ul/100ml), culture at 37°C until OD ₆₀₀ = 0.5-0.6, collect 1ml of bacterial liquid as a control; Bit 0.2g/L; 4 hours after the induction culture, the bacterial liquid was collected for purification.

1.1.2.8 Purification of recombinant protein of giant panda ribosomal protein subunit gene RPL23a

1.1.2.8.1 Assembling the chromatography column

Mix the filler evenly and add it to the chromatographic column, and let it stand at room temperature for 10 minutes. After the gel and the solution are separated, let the ethanol flow out slowly by gravity. Add 5 times the column volume of deionized water to the packed column to rinse the ethanol, then equilibrate the column with 8 times the column volume of Binding Buffer, and load the sample after the equilibration.

1.1.2.8.2 Purification of protein

After collecting the cells, add 1-5ml of bacterial lysate per 100mg of cells (wet weight), and ultrasonically lyse the cells. Centrifuge, discard the supernatant, and resuspend the pellet in Soluble Binding Buffer. Ice-bathed for 1 hour to dissolve the inclusion bodies. Centrifuge at 10,000×g for 20 minutes, and filter the supernatant through a filter membrane with a pore size of 0.45 μm. Load the protein solution onto the column at a flow rate of 10 times column volume/hour. Wash the column with 15 column volumes of Inclusion Body Binding Buffer and collect the flow-through peaks. Use 5 column volumes of Inclusion Body Elution Buffer to elute, and collect the eluted peaks. The eluates were combined, and the purity of the recombinant protein of the giant panda ribosomal protein subunit gene RPL23a was detected by DS-PAGE. The concentration of recombinant protein was measured with Bradford Protein Concentration Assay Kit (Mbchem ^TM M2031).

1.1.2.9 Cell culture

Human laryngeal cancer Hep-2 cell line was incubated at 37°C with RPMI-1640 complete culture solution containing 10% inactivated fetal bovine serum (fetalbovine serum, FBS) 100 U/ml penicillin, 100 μg/ml streptomycin, pH 7.4 , 5% CO ₂ incubator for cultivation.

1.1.2.9.1 MTT method to detect cell viability

Take the cells in the logarithmic growth phase, digest with trypsin and adjust the number of cells to 1× ¹⁰⁵ cells/mL, inoculate in a 96-well plate, the volume of cell fluid in each well is 200 μL, put them in a CO ₂ incubator, and incubate at 37 ℃, 5% CO ₂ and saturated humidity conditions, incubate until the cell monolayer covers the bottom of the hole (96-well flat bottom plate), and then add the giant panda ribosomal protein subunit RPL23a recombinant protein to make the final concentrations of 0, 7.4, 2.96, 1.48, 0.74, 0.37, 0.185, 0.148 μg/mL, 6 replicate wells were set up for each concentration, and the blank group was the RPMI1640 culture solution without cells, put it into the _CO2 incubator for 24 hours and then take out the 96-well plate, Add 10 μL of MTT (5 mg/mL) solution, continue to incubate for 4 hours, then add 150 μL of dimethyl sulfoxide (DMSO) and shake for 10 minutes. After the blue-purple crystals are completely dissolved, use a microplate reader to measure each concentration at a wavelength of 490 am. The absorbance (OD) value of the hole is used to calculate the inhibition rate (%) of the action of the giant panda ribosomal protein subunit RPL23a recombinant protein on Hep-G2 cells under different concentrations. The formula is as follows: cell growth inhibition rate=(1-A _{experimental group} /A _{Control group} ) × 100%.

1.1.2.9.2 Morphological observation

The 96-well plate was observed under an inverted microscope, and the morphological changes of cells at different concentrations were recorded by taking pictures.

2 results

2.1 The cDNA sequence and encoded amino acid sequence of the giant panda ribosomal protein subunit RPL23a gene

The obtained RT-PCR amplification product was electrophoresed with 1% agarose gel, and a clear specific amplification band of about 500 bp was seen. The result of sequencing the recombinant plasmid showed that the obtained sequence was 506bp in length (SEQ ID NO: 5)). Using the Blast search software in the NCBI database to search for gene homology, it was found that the sequence has a high homology with the cDNA sequence of the RPL23a gene of humans and other animals, which proves that the cloned sequence is the giant panda RPL23acDNA sequence. The ORF of the sequence is 471bp, the start codon is ATG, and the stop codon is TAA. It encodes 156 amino acid residues (SEQ ID NO: 6), and the molecular weight of the giant panda ribosomal protein subunit RPL23a is predicted to be 17.72KDa. The isoelectric point is 10.44.

2.2 Giant panda ribosomal protein subunit gene RPL23a recombinant protein

The obtained giant panda ribosomal protein subunit gene RPL23a recombinant protein sequence (190 amino acid residues), (SEQ ID NO: 7). The molecular weight of the recombinant protein is 21.265KDa; the isoelectric point is 10.57;

2.3 The effect of recombinant protein of giant panda ribosomal protein subunit gene RPL23a on the growth of Hep-2 cells

MTT method was used to detect the effects of different concentrations of giant panda ribosomal protein subunit gene RPL23a recombinant protein on the growth activity of Hep-2 cells under 24 hours. It can be seen from Figure 1 that the recombinant protein of the giant panda ribosomal protein subunit gene RPL23a has a time- and dose-dependent inhibition rate on the growth of Hep-2 cells. It is found that the effect at low concentration is better than that at high concentration, and the recombinant protein of giant panda ribosomal protein subunit gene RPL23a has the best growth inhibition rate on Hep-2 cells when the concentration is 0.185 μg/ml, which can reach 36.31%. (see picture 1).

2.4 Effects of giant panda ribosomal protein subunit gene RPL23a recombinant protein on the morphology of Hep-2 cells

Use an inverted microscope to observe the morphological changes of Hep-2 cells treated with different concentrations of giant panda ribosomal protein subunit gene RPL23a recombinant protein (0, 7.4, 2.96, 1.48, 0.74, 0.37, 0.185, 0.148 μg/mL) for 24 hours (Figure 2) . It can be seen from Figure 2 that under the treatment time of 24 hours, compared with the control group, as the concentration of the recombinant protein of the giant panda ribosomal protein subunit gene RPL23a decreased, the cells appeared round, the intracellular particles increased, and the cells formed into clusters , shedding or even cracking into fragments (Figure 2), and the recombinant protein of giant panda ribosomal protein subunit gene RPL23a had the greatest impact on the morphology of Hep-2 cells when the concentration was 0.185 μg/ml.

It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should belong to the protection scope of the appended claims of the present invention.

Claims (2)

1. Giant panda ribosomal protein subunit gene RPL23a recombinant protein, the amino acid sequence of the recombinant protein is defined by SEQ ID NO:7. 2. The application of the giant panda ribosomal protein subunit gene RPL23a recombinant protein according to claim 1 in the preparation of anticancer drugs.

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