CN111592585A

CN111592585A - Application of UL16 protein in preparation of drugs for promoting cell mitochondrial function

Info

Publication number: CN111592585A
Application number: CN201811537353.XA
Authority: CN
Inventors: 孙晗笑; 张倩; 利时雨
Original assignee: Guangzhou Traceable Biotechnology Co ltd
Current assignee: Guangzhou Traceable Biotechnology Co ltd
Priority date: 2018-12-15
Filing date: 2018-12-15
Publication date: 2020-08-28

Abstract

The invention discloses application of UL16 protein in preparation of small molecule drugs for promoting in vitro differentiation of embryonic stem cells and application of stem cell transplantation in treatment of nervous system degenerative diseases. According to the invention, through constructing the UL16 plasmid transfected host cells, it is found that UL16 can promote the aerobic oxidation and oxidative phosphorylation of glucose, and activate the cell productivity way, so that the intracellular ATP content is increased, and the mitochondrial function is improved. Finally, the UL16 expression plasmid is constructed to transfect the mouse embryonic stem cells, and the mitochondrial function state and the energy metabolism are found to be enhanced, so that the stem cell differentiation can be promoted. Realizes clinical treatment of cells in a differentiation defect state and cells with low differentiation rate, and is used for treating diseases or pathological and physiological processes related to mitochondrial dysfunction or low of cells.

Description

Application of UL16 protein in preparation of drugs for promoting cell mitochondrial function

Technical Field

The invention belongs to the field of protein screening and application mechanisms, and particularly relates to a protein UL16 with functions of enhancing mitochondrial function state and increasing stem cell differentiation efficiency and application thereof.

Background

Embryonic Stem Cells (ESCs) provide a seed cell source for cell replacement therapy of various metabolic liver diseases due to the capability of self-renewal, high proliferation and multidirectional differentiation, so that the ESCs become a hotspot of research make internal disorder or usurp, but the low efficiency of induced differentiation is a key problem which is difficult to solve. In vitro, mouse cells can be directionally differentiated into almost all types of adult cells; human cells can also successfully differentiate into various types of cells such as neurons, hepatocytes, endothelial cells, cardiomyocytes, pancreatic cells, and hematopoietic cells. In vitro studies show that stem cells from different sources can be differentiated towards liver cells, express surface markers related to the liver cells, have some liver cell functions, but are low in differentiation efficiency. Many normal cells increase oxygen and energy consumption during cell division, during which the cell will rest in G1 without any DNA damage if ATP synthesis is insufficient. Down-regulation of mitochondrial oxidative phosphorylation has been reported to cause premature senescence in somatic cells and mice; more interestingly, the research on the mitochondria and energy metabolism of adult/Embryonic Stem Cells (ESCs) by a plurality of researchers recently found that the functions of the mitochondria may affect the proliferation, differentiation potential, survival period and the like of normal stem cells. Experiments prove that human embryonic stem cells are implanted into injured tissues of aged mice, the embryonic stem cells have an inherent anti-aging effect, and a substance capable of generating soluble protein is an anti-aging signal in a MAPK (mitogen-activated protein kinase) path.

Mitochondria are involved in a variety of cellular signaling pathways and metabolic pathways, and play several key functions in cellular metabolism and antiviral signaling pathways, including their central role in ATP production, while there are many reports that elucidate the role of mitochondria in the maintenance of stem cell pluripotency. Mitochondrial dysfunction can thus accelerate aging in mammals and trigger many of the associated pathophysiological deficiencies. Viruses are also no exception to rely on host mitochondria-associated metabolic pathways, such as HSV-1 relies on the host cell's metabolic network to supply energy and macromolecular precursors for viral replication. Can activate glycolysis pathway of host cell by increasing activity of fructose-6-phosphate kinase (PFK-1), so as to increase glucose uptake of host cell and intracellular ATP content of cell. From this, it is clear that the viral proteins are closely related to mitochondria.

We have found that such a mitochondrially localized viral protein UL16, as a mitochondrial stimulating factor, plays an important role in combating senescence caused by mitochondrial dysfunction and has applications in improving the pathophysiological processes associated with mitochondrial dysfunction. This opens up new chapters for later studies of viral proteins as beneficial embryonic stem cell clinical therapies. Therefore, it becomes one of the important research directions in the future to discuss the immunogenicity of UL16 protein, the ability of inducing the energy homeostasis of the organism, the target of preventing or treating new drug action, etc.

Disclosure of Invention

The invention discovers the function of herpes virus protein UL16 in mitochondria and researches the action mechanism of the herpes virus protein UL16 so as to ensure that the herpes virus protein UL16 plays a role in treating diseases.

The invention discovers that the virus protein UL16 can be applied to preparing medicines for treating anti-aging, mitochondrial dysfunction or metabolic syndrome.

The invention also finds that UL16 can be applied to the differentiation process of embryonic stem cells to promote mitochondrial energy metabolism.

This chapter successfully constructs the prokaryotic expression plasmid pET-31b (+) -UL16 of UL16 gene, and transforms into (DE3) expression host bacteria. Western blotting experiments show that the recombinant protein has the same molecular weight as the predicted molecular weight, and is mainly expressed in an insoluble inclusion body form; immunoblotting with rabbit antibody to obtain specific band; the purified renatured recombinant protein is used for immunizing a New south China white rabbit to prepare a rabbit anti-polyclonal antibody, the prepared polyclonal antibody can be specifically identified with the recombinant protein, the recombinant protein is proved to have good immunogenicity and reactogenicity, and the prepared polyclonal antibody can be used for the subsequent gene function research. Compared with the prior art, experiments show that the invention enhances the aerobic oxidation of cell glucose, improves the mitochondrial respiration and regulates and controls the ATP energy metabolism pathway, thereby realizing the treatment and prevention of various metabolism-related diseases such as cell aging, mitochondrial dysfunction, metabolic syndrome and the like.

Drawings

FIG. 1 shows the results of UL16 gene identification in example 1.

FIG. 2 is the electrophoresis chart of UL16 protein in example 1.

FIG. 3 shows ATP levels after transfection of HEK293T cells with the recombinant plasmid of example 2.

FIG. 4 shows the effect of UL16 gene on glucose metabolizing enzyme activity as a result of co-immunoprecipitation in example 2.

FIG. 5 shows the effect of lactate levels of UL16 gene of example 2.

FIG. 6 is a graph of the determination of glucose consumption levels for UL16 transfection of example 2.

FIG. 7 is a graph showing the measurement of the effect of transfection of recombinant plasmid on the number of mitochondria in example 2

FIG. 8 shows mtDNA levels after transfection of embryonic stem cells with the recombinant plasmid of example 2.

Detailed Description

The present invention will be described in further detail with reference to examples.

Example 1

1. Experimental methods and materials

UL16 expression plasmid pET-31b (+) -UL16 for HSV-1, expression vector pET-31b (+) from Invitrogen, and plasmid pFLAG-CMV-2 from Sigma. Herpes simplex virus F strain (HSV-1F), strain DH5 alpha receptor bacteria and cells are all preserved in the laboratory.

Primer design and Synthesis

Based on the sequence of HSV-1UL16 gene queried at NCBI, primers UL16-for and UL16-rev were designed for amplification of HSV-1UL16 using Primers5.0 software, which was synthesized by Shanghai Producer.

TABLE 1-1 primers used for construction

1.2 Virus amplification and template preparation

And (3) taking the single-layer HEK293T cells for culturing HSV-1 virus, and collecting and treating virus liquid when about 75% of cells are diseased, so as to finally obtain HSV-1 genome and serve as a target gene amplification template. The specific operation steps are as follows:

(1) taking a monolayer of HEK293T cells, discarding the original cell culture solution, repeatedly washing with PBS buffer solution for 3 times, and sufficiently removing dead or nonadherent cells.

(2) HSV-1 virus was removed from the freezer at-80 ℃ and lysed.

(3) Add 200. mu.l of virus to the cells, mix well and place in the incubator. Shaking every 15-20 min to make virus completely adsorb cells.

(4) After about 2 hours the virus was able to complete the infection of the cells, 4 ml of fresh cell culture was added and incubated overnight in a 5% CO2 incubator at 37 ℃.

(5) The next day early, when cell morphology was observed under an inverted microscope, about 75% of the cells became rounded and the refractive index increased, indicating that the virus had successfully infected the cells.

(6) After being fully and uniformly blown by a suction pipe, 200 mul of each pipe is respectively arranged in a freezing storage pipe and stored at minus 80 ℃ for standby.

(7) Taking a proper amount of virus liquid, boiling in water bath at 100 ℃ for 10min, then centrifuging at 12000rpm for 5min, and obtaining the target gene amplification template through centrifugal precipitation.

Cloning of genes

The primers UL16-for and UL16-rev (shown in Table 1-1) are used for amplifying HSV-1UL16 target genes, and PCR products are purified and recovered. The specific operation steps are as follows:

(1) the PCR reaction system is as follows:

10×PCR Buffer	5.00μl
		DNA polymerase	0.25μl
dNTP(2.5mM)	2.00μl
		MgCl₂	1.50μl
UL16-for(10μM)	1.00μl
		UL16-rev(10μM)	1.00μl
Form panel	4.00μl
		ddH₂O	12.75μl
Total of	25.00μl

Setting PCR conditions: 5min at 94 ℃, 1min at 55 ℃, 30s at 72 ℃ for 1min, and 30 cycles; 10min at 72 ℃. After the PCR is finished, the PCR product is stored at 4 ℃ and separated and identified by 1% agarose gel electrophoresis.

Recovery and purification of the product

The PCR products were verified by nucleic acid electrophoresis using 1% agarose gel. And recovering and purifying the target fragment by using the gel recovery kit. The specific operation steps are as follows:

(1) after the electrophoresis is finished, the agarose gel block containing the bright band of the DNA fragment is cut off under UV irradiation with a sterilized clean rubber cutter. When cutting the gel, the empty agar block is cut as much as possible, and the gel cutting speed is high, so that excessive DNA loss is prevented.

(2) To convert the volume of about 1. mu.l gel per 1 mg gel, 3 times the volume of Buffer W1 was added to the EP tube, and the tube was incubated in a water bath at 65 ℃ for 10min, all with shaking until the gel was completely dissolved.

(3) Then, half the volume of Buffer W2 as much as the amount of Buffer W1 was added to the EP tube, mixed well, centrifuged at 12000rpm for 30s, the supernatant was discarded, and the operation was repeated once.

(4) Rinsing twice with washing solution, centrifuging at 12000rpm for 1min, and discarding the supernatant.

(5) Adding 35 μ l of Eluent, standing for 5min, centrifuging at 12000rpm for 2 min, and eluting the DNA to obtain the purified target DNA.

Preparation of expression plasmid pET-31b (+) -UL16

1.5.1 in vitro recombination of fragments of interest and cloning vectors

(1) UL16-for and UL16-rev are used as upstream and downstream primers, pHUL16 plasmid is used as a template, independent PCR processes are respectively carried out, 1% agarose gel electrophoresis identification is carried out on PCR products, and purified DNA is recovered.

(2) Meanwhile, the pET-31b (+) empty plasmid is cut by EcoR I and BamH I to prepare pET-31b (+) vector large fragment.

(4) Products recovered after double enzyme digestion of UL16 and mutant UL16 are respectively mixed with pET-31b (+) vector large fragments, and pET-31b (+) -UL16 expression plasmids are obtained under the action of T4 DNA ligase.

Preparation of competent cells

(1) The DH5 alpha strain frozen in the laboratory was inoculated into LB plate and cultured at 37 ℃ for 16 h.

(2) A single colony was selected and inoculated into 50 ml of LB liquid medium and cultured at 37 ℃ until the OD value became about 0.5 (range 0.4-0.6).

(3) Transferring 25 ml of the bacterial solution into a precooled 50 ml EP tube, standing on ice for 30min, cooling the culture to 0 ℃, centrifuging at 4 ℃, 4000 rpm for 10min, discarding the supernatant, and recovering the thalli.

(4) 5 ml of pre-cooled 0.1 mol/L CaCl2 was added, each pellet was resuspended and placed on an ice bath for 30 min. Centrifuging at 4 deg.C and 4000 rpm for 10min, removing supernatant, and recovering thallus.

(5) 1 ml of 0.1 mol/L CaCl2 (containing 15% glycerol) pre-cooled with ice was added to resuspend the bacterial pellet.

(6) Cells were split into small portions, 100 ul/portion, frozen at-80 ℃ for use on ice.

Transformation of recombinant plasmids

(1) Taking a proper amount of DH5 alpha competent cells, adding 9 mu l of products connected with reactants, mixing uniformly, and carrying out ice bath for 30 min;

(2) placing into 42 deg.C water bath, and heat shocking for 2 min; immediately transferring to an ice water bath for cooling for 1 min;

(3) cooling, adding 400 μ l LB liquid culture medium, culturing at 37 deg.C and 180 rpm under shaking for 1 hr to activate

(4) Spreading on LB solid culture medium containing X-gal, IPT, Amp, placing in biochemical incubator at 37 deg.C for 15min, and performing inverted culture for 16-20 h after the bacterial liquid is completely absorbed.

Identification and extraction of plasmids

White colonies were picked and inoculated into 5 ml LB liquid medium (with ampicillin resistance) and cultured overnight, according to the plasmid mini-extraction kit, as follows:

(1) 2ml of the bacterial culture was centrifuged at 12000rpm for 1min, the supernatant was discarded, and 250. mu.l of solution 1 (containing RNaseA) was added thereto, followed by shaking to suspend the pellet thoroughly.

(2) Add solution 2 to EP tube, turn 6-8 times up and down. Adding the solution 3, turning over for 6-8 times, and mixing. Centrifuge at 12000rpm for 10min and transfer the supernatant to another EP tube.

(3) Adding the supernatant into adsorption column, standing at room temperature for 2 min, centrifuging at 12000rpm for 1min, discarding the waste liquid, and replacing the adsorption column into the collection tube.

(4) Adding 750 μ l of column washing solution into the elution column, centrifuging for 1min (12000 r/min), discarding the solution, pouring 250 μ l of column washing solution, centrifuging for 5min (12000 r/min), and discarding the solution.

(5) Add 50. mu.L of deionized water to the column, let stand for 2 min, centrifuge to collect the liquid, and store at-20 ℃ for use.

Liposome transfection

The following procedure was performed according to the Lipofectamine 2000 Lipofectamine kit:

(1) cells were seeded at 0.5-2X 105 cells per well in 24-well plates in 500. mu.l DMEM medium without antibiotics and serum, and cells were as long as 90-95% confluent upon transfection.

(2) Plasmid DNA was diluted with 50. mu.l of Opti-ME I serum-free medium and mixed gently. An appropriate amount of Lipofectamine 2000 was diluted in 50. mu.l Opti-ME I medium and incubated for 5min at room temperature.

(3) The DNA diluted in the first two steps was mixed with Lipofectamine 2000 (to make the total volume 100. mu.l), gently mixed, and left at room temperature for 20 minutes.

(4) 100 μ l of transfection solution was added to each well of cells and shaken gently.

(5) The medium was changed after 4-6 h of transfection at 37 ℃ with 5% CO2 and cells were lysed 24 h later in preparation for detection of gene expression.

Identification of the protein of interest

The expression condition of HSV-1UL16 protein is detected by Western blotting, and the specific operation steps are as follows:

(1) preparing glue: preparing 12% separation glue, injecting into the gap between the glass plates to a position 1.5 cm away from the upper edge, and adding appropriate amount of 75% ethanol into the upper layer; pouring out the upper layer liquid after solidification, injecting 5% concentrated glue, inserting a comb, and naturally drying.

(2) Loading: the protein sample is bathed in water at 100 ℃ for 3 min, the newly configured electrophoresis solution is poured into the electrophoresis tank, 8 mu l and 5 mu l of protein maker are respectively added into two lanes, the volume is filled to 10 mu l by 1 times of protein loading buffer solution, 10 mu l of sample is respectively added into the other lanes, and the electrophoresis is carried out at constant voltage of 50V.

(3) Film transfer: after electrophoresis is finished, cutting filter paper of 11 cm multiplied by 8 cm and a PVDF film of 0.22 mu m with proper size, activating for 5min by using methanol, assembling a transfer printing interlayer according to the 'sponge-6 layers of filter paper-gel-PVDF film-6 layers of filter paper-sponge', transferring to a film transferring groove after the assembly of a splint, and transferring for 60 min at a constant current of 200 mA.

(4) Incubation of the antibody: after the membrane transfer is finished, washing the membrane for 5min by TBS (TBS), sealing by 5% skimmed milk powder for 1 h, washing the membrane for 3 times by TBST (TBST), adding an anti-diluent for incubation at 4 ℃ overnight; washing the membrane with TBST solution for 3 times, each time for 5min, adding secondary antibody diluent, and incubating at room temperature for 1 h.

(5) Luminescence detection: washing the membrane with TBST solution, adding luminescent solution, incubating for 3 min, sucking out the luminescent solution with filter paper, placing the membrane and PVDF membrane into a tabletting box, tabletting for 1min, taking out the membrane, fixing for 30s, washing with water, oven drying, and scanning.

Example 2 transfection of pET-31b (+) -UL16 into cells and intracellular function Studies

1. Experimental Material

HEK293T cells were also cryopreserved by this laboratory.

Experimental methods

1.2.1 ATP assay

According to the operation of the ATP content determination kit instruction:

(1) the reagent to be used was dissolved in ice, and the ATP standard solution was diluted to concentrations of 0.1, 1 and 10. mu.M/L using an ATP assay lysate to prepare a standard curve.

(2) Add 200. mu.l lysis solution into the cell culture dish, blow repeatedly until fully lysis, centrifuge at 12000 rmp at 4 ℃ for 10min, take the supernatant.

(3) Diluting the ATP detection working solution, repeating the steps for 3 times for each sample, adding 100 mul of ATP detection working solution into each monitoring hole, standing at room temperature for 5min, consuming local ATP, adding 100 mul of samples to be detected or standard samples into the detection holes, fully mixing, and immediately detecting the CMP value by using a bioluminescent instrument at intervals of 2 s. And calculating the ATP concentration in the sample to be detected by using the measured standard curve according to a calculation formula.

1.2.2 pyruvate kinase Activity assay

According to the instructions of the pyruvate kinase kit:

(1) taking 400 μ l of cell extract, ultrasonically breaking cells (power 20%, ultrasonic for 3 s, interval 10 s, repeating for 30 times), centrifuging at 8000 g and 4 deg.C for 10min, and storing supernatant for use.

(2) And mixing the reagent IV and the reagent V in the kit uniformly, carrying out water bath at 37 ℃ for 5min, adding 30 mu l of sample, starting timing, and recording the initial absorbance A1 at the wavelength of 340 nm for 20 s.

(3) And then, quickly and accurately reacting the mixture in a water bath of 37 ℃ in a cuvette for 2 min, quickly taking out the cuvette, wiping the cuvette by using a piece of mirror wiping paper, carrying out color comparison at 340 nm, recording the absorbance A2 at 2 min of 20 s, and calculating the delta A = A1-A2.

(4) PK enzyme activity calculation formula: PK (U/10)⁴cell) = total reaction volume ÷ reaction time ÷ NADPH extinction coefficient ×△ a ÷ viable cell density.

1.2.3 detection of fructose-6-phosphate kinase Activity

The instructions for the kit were as follows:

(1) the cell processing method is the same as step one in 3.3.7.

(2) Mu.l of PFK working solution, 30. mu.l of sample, six 5. mu.l of reagent and seven 5. mu.l of reagent are sequentially added into a1 mL cuvette, and the absorbance of the sample is A1 when the sample is recorded at a wavelength of 340 nm for 20 s.

(3) Then, the mixture was taken out and wiped dry with a paper mirror for 10min at 37 ℃, and the absorbance at 20 s for 10min was measured as A2 by colorimetry at 340 nm, and Δ A = A1-A2 was calculated.

(4) PFK enzyme activity calculation formula: the formula is calculated as in 4.3.3.

1.2.4 measurement of glucose consumption

(1) In the same order of magnitude of 1 × 10⁵Placing the serum-free monolayer adherent cells into a 6-hole plate, adding 2ml of serum-free stem cell culture solution for culture, placing 1 × 105A 549 cell line cells with the same order of magnitude into the 6-hole plate, respectively adding 2ml of serum-free stem cell culture solution and 10% of fetal bovine serum DMEM culture medium, and performing differentiation culture under the conditions of 37 ℃, 5% CO2, 5% atmosphere and saturated humidity;

(2) after 24 hours and 48 hours of the culture, the blood glucose concentrations of the cell culture solutions of the respective groups were measured at the beginning and at 24 hours and 48 hours;

(3) glucose concentration of the culture solution was measured by glucose oxidase method (according to the glucose measurement kit instructions):

adding 100 mul of each of the reagents R1 and R2 in the kit into each well of a 96-well plate;

adding culture solution to be detected, standard substance (glucose concentration 5.5mmol/L) and distilled water (blank tube) into the holes respectively by 2 mul, mixing well, and 15min at 37 ℃; in an enzyme-linked immunosorbent assay (ELSA), at the wavelength of 505nm, the blank tube is zeroed, and the light absorption values (A) of the sample tube and the standard tube are read; the sample glucose concentration is sample tube A/standard tube A multiplied by standard tube glucose concentration;

(4) the initial glucose concentration was subtracted from the glucose concentration at each time point in the cell culture medium of each group, and the difference was the glucose consumption of the cells of each group.

1.2.5 mitochondrial number detection

(1) Placing ESCs cells 1 × 105 in a cover glass six-hole plate, adding DMEM medium containing 10% fetal calf serum, culturing at 37 deg.C under 5% CO2 and 5% atmosphere and saturated humidity for 3 days, and making cell slide;

(2) removing culture solution, adding 2ml of DMEM medium containing Hoechst 33342 (staining nuclei) at 5 μ g/ml, and incubating at 37 deg.C in dark for 100 min;

(3) the medium was aspirated and washed 3 times with PBS;

(4) adding 2ml DMEM medium containing 100nM Mito-Tracker Green (mitochondria independent mitochondrial membrane potential Green fluorescent probe), and incubating at 37 deg.C in dark for 30 min;

(5) discarding the staining solution, washing with PBS or culture solution for 2-3 times to perform fluorescence detection;

(6) taking out the cover glass, and sealing the cover glass by using an anti-fluorescence quenching sealing liquid;

(7) and (5) observing by using a laser confocal microscope. During detection, the maximum excitation wavelength of the Mito-Tracker Green is 488nm, and the maximum emission wavelength is 525 nm; hoechst 33342 has a maximum excitation wavelength of 352nm, maximum emission wavelengths of 530 nm (blue) and 630 nm (red);

(8) a549 cells are divided into plasmid transfected cells and non-transfected cells according to the light staining condition of cell nuclei, and the distribution morphology proportion of mitochondria and the fluorescence intensity of mitochondria (representing the number of mitochondria) in the two cells are compared.

1.2.6

2. experimental results

2.1 construction result and identification of plasmid pET-31b (+) -UL16

The NCBI inquiry shows that the size of the normal HSV-1UL16 gene is 1122bp, primers UL16-for and UL16-rev are utilized, the constructed HSV-1UL16 eukaryotic expression vector plasmid pHUL16 is used as a template, PCR amplification products can be observed through electrophoresis to obtain two amplified specific target bands (shown in figure 1) of about 1122bp, and the specific target bands are consistent with expected results before experiments.

2.2 Western blot to verify the expression of recombinant proteins

And inoculating the transformed expression bacteria containing the positive recombinant plasmid pET-31b (+) -UL16 into a culture medium containing the expression bacteria for induced expression, and carrying out induced expression on the empty vector pET-31b (+) transformed bacteria. The results show that: the empty vector transformation bacterium induces and expresses a protein band with the size being about the same as that of the tag protein, and the bacterium containing the recombinant expression plasmid pET-31b (+) -UL16 finds a target band at about 41 kDa. As shown in FIG. 2, it was confirmed that the plasmid pET-31b (+) -UL16 was normally expressed in cells, specifically identified from the protein level.

2.3 Effect of pET-31b (+) -UL16 on the oxidative pathway of cellular glucose

Glycolysis and the TCA cycle are central to central carbon metabolism in mammalian cells. Through both pathways, glucose is oxidized, producing energy in the form of NADH and ATP, or converted to precursors of amino acids, lipids, and nucleotides.

2.3.1 Effect of pET-31b (+) -UL16 transfection on ATP production by cells

After HEK293T cells are transfected by pET-31b (+) -UL16, the intracellular ATP level is obviously increased. pET-31b (+) -UL16 transfected cells had approximately 2-fold higher levels of ATP than cells transfected with WT and empty vector at 48 h, and slightly increased at 24 h (FIG. 3).

2.3.2 Effect of pET-31b (+) -UL16 transfection on cellular glucose metabolizing enzyme Activity

The key rate-limiting enzymes of glycolytic pathway regulation are fructose-6-phosphate kinase (PFK-1) and Pyruvate Kinase (PK). pET-31b (+) -UL16 significantly increased PFK-1 and PK activity 48 h after transfection (FIG. 4).

2.3.3. Effect of pET-31b (+) -UL16 transfection on cellular lactate production

The above results demonstrate that pET-31b (+) -UL16 enhances glucose metabolism, and to further demonstrate the increased aerobic oxidation of glucose, lactate levels were measured by the kit. The results indicate that pET-31b (+) -UL16 transfected cells showed significantly lower lactate levels than the empty vector transfected and wild type cells (FIG. 5).

2.3.4 Effect of pET-31b (+) -UL16 transfection on cellular glucose uptake

HEK293T cells transfected with pET-31b (+) -UL16 were cultured in serum-free medium and glucose consumption was determined to be lower than that of the control group after 48 hours of glucose consumption as measured by serum-free monolayer adherent cells and the A549 cell line was transfected by the test recombinant plasmid (FIG. 6).

2.4 Effect of pET-31b (+) -UL16 transfection on the number of mitochondria in cells

The fluorescence intensity of mitochondria Mito Tracker Green of the embryonic stem cells transfected with pET-31b (+) -UL16 plasmid and the cells of a control group has no significant difference through the observation of a laser confocal microscope. Indicating no significant difference in the number of intercellular mitochondria before and after cell transfection (fig. 7).

2.5 pET-31b (+) -UL16 transfection Effect on mt DNA copy number of ESCs

Although there was no significant difference in the number of mitochondria between cells before and after pET-31b (+) -UL16 transfection, the mtDNA copy number (Cox I/β -actin value) of ESCs cells was increased compared to that before transfection (FIG. 8).

pET-31b (+) -UL16 eukaryotic expression vectors are successfully constructed and transfected into HEK293T cells; fluorescence microscopy and Western blot results show that the UL16 protein is stably expressed in HEK293T cells. Then, the ATP content in cells and the enzyme activities of PFK-1, PK and LDH in the glucose aerobic oxidation process are respectively detected by using related kits, and the fact shows that UL16 increases the glucose metabolism and the ATP content of the cells; in addition, the oxygen consumption and the lactic acid level of UL16 transfected cells are detected, and the oxygen consumption of the cells is increased, and the lactic acid level is reduced. No significant difference in mitochondrial number was found after UL16 transfection of embryonic stem cells, and the copy number of mitochondrial DNA after transfection was increased as shown by increased activity of enzymes such as COX 1. These results above indicate that UL16 transfected HEK293T cells increased aerobic oxidation of cellular glucose, promoting energy metabolism during differentiation of embryonic stem cells.

Claims

1. An expression plasmid of herpesvirus UL16 gene, and active fragment of UL16 gene or its related nucleic acid and protein.

2. Use of the plasmid expressing UL16 protein according to claim 1 in the preparation of a medicament for treating mitochondrial dysfunction or promoting stem cell differentiation.

3. The plasmid expressing UL16 protein of claim 1, wherein the transfection of UL16 gene promotes mitochondrial energy metabolism, can be used for the preparation of a medicament for the prevention of mitochondrial dysfunction.

4. The plasmid expressing UL16 protein of claim 1, which can be used for preparing a differentiation-promoting drug for stem cells, wherein the differentiation-promoting drug comprises UL16 protein as an active ingredient in an effective dose to promote the perfection of an in vitro differentiation-inducing scheme of stem cells so as to promote the clinical application of stem cell transplantation.

5. The plasmid expressing UL16 protein of claim 1, wherein the efficiency of stem cell differentiation is promoted by enhancing mitochondrial function in differentiation.

6. The plasmid of claim 1 for expressing UL16 protein, wherein the plasmid can be used in drugs for regulating the processes of tumor generation, proliferation and metastasis by influencing the function of cell mitochondria.

7. The plasmid of claim 1 for expressing UL16 protein, which is characterized by being capable of treating and preventing various metabolic-related diseases such as cell aging, mitochondrial dysfunction and metabolic syndrome.