阅读说明：本技术 一种提高重组柯萨奇病毒溶瘤作用的方法 (Method for improving recombinant coxsackie virus oncolytic effect ) 是由 蔡立刚 曹雪芹 刘志毅 于 2020-12-29 设计创作，主要内容包括：本发明涉及及生物制品制备技术领域,具体公开了一种提高重组柯萨奇病毒溶瘤作用的方法,病毒经细胞培养纯化后,将细菌脂多糖(LPS)作为协同剂加入病毒体系可以有效提肿瘤细胞出现病变效应,从而提升柯萨奇病毒的溶瘤作用,扩大柯萨奇病毒用于治疗肿瘤的用途。(The invention relates to the technical field of biological product preparation, and particularly discloses a method for improving the oncolytic effect of a recombinant coxsackie virus.)

Use of LPS in the preparation of an oncolytic medicament comprising coxsackievirus CVB 3-a.

2. Use according to claim 1, wherein the concentration of LPS is between 50 and 1000 μ g/ml.

3. Use according to claim 1, wherein the LPS concentration is 50 μ g/ml, 100 μ g/ml, 500 μ g/ml, 1000 μ g/ml.

4. Use according to claim 1, wherein the LPS concentration is 500 μ g/ml.

5. The use of claim 1, wherein the coxsackievirus CVB3-a is administered at a dose of 10⁹PFU。

Technical Field

The invention belongs to the technical field of biological product preparation, and particularly relates to a method for improving the oncolytic effect of a recombinant coxsackie virus.

Background

Coxsackie Virus (CV) was first obtained in stool samples by Gillbert Dalldorf, 1948, in search for a cure for poliomyelitis disease, and was named after its discovery in the new york coxsackie town. Like other types of enteroviruses, coxsackieviruses can be transmitted via the fecal-oral and oral-oral routes. Among other things, fecal contamination of water, food, and soil from contact with infected persons can lead to fecal-oral routes of transmission. Generally, infants are mostly recessive infected; and the detoxification time of children under 5 years old, especially children under 3 years old can be as long as one month. After the virus infects human body, diseases such as upper respiratory tract infection, acute myocarditis and the like can be caused, and serious threat is caused to the health of infants.

The coxsackievirus B group often causes mild cold-like illness and sometimes can cause severe lesions of the heart, pancreas or central nervous system, especially in newborns, toddlers and immunodeficient adults. Such as intrauterine infection or perinatal infection in late pregnancy can lead to serious fatal diseases such as neonatal myocarditis, meningitis, encephalitis, hepatitis and the like.

With the development of whole genome sequencing technology, sequencing of the genome amino acid sequence of more and more CVB3 strain has been completed. It has been found that certain base changes in the genome of different strains of CVB3 virus result in completely different structures and functions of the proteins they encode, and thus in different virulence. The strains CVB3/20, CVB328, CVB3AS and CVB/Nancy cause myocarditis and pancreatitis, the virus CVB3CO causes pancreatitis only, while CVB3GA, CVB3/0 do not cause disease.

Chinese patent publication CN103981152A discloses a coxsackievirus, which is a coxsackievirus B group 3 type variant, wherein the 2690 site of the genome of the coxsackievirus is adenine and the 3231 site of the genome is guanine. The Coxsackie virus has obvious cytolytic capacity and selection specificity, and can be applied to preparation of antitumor drugs.

Bacterial Lipopolysaccharide (LPS) is a component of the outer wall of the cell wall of gram-negative bacteria, and is a substance (glycolipid) composed of lipids and polysaccharides. Lipopolysaccharides are present in the extracellular lipids covering the outer membrane (lipid bilayer) of the whole body. What is considered to play the most important role in the manifestation of the physiological activity of LPS is the lipid a moiety, which alone may manifest its physiological role. LPS as a component of the cell wall of enterobacteria may have a synergistic effect on enterovirus-like viruses, however, this has not been studied in the prior art.

Therefore, it is necessary to research the anti-tumor effect of LPS on coxsackie virus aiming at the possible synergistic effect of LPS, so as to expand the application range of LPS and improve the anti-tumor effect of coxsackie virus in the prior art.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for improving the oncolytic effect of a recombinant coxsackie virus, thereby expanding the application range of LPS and improving the oncolytic effect of the coxsackie virus in the prior art.

In order to solve the problems, the scheme of the invention is as follows:

the invention provides an application of LPS in preparing an oncolytic medicament, wherein the medicament comprises coxsackie virus CVB 3-A.

Further, the concentration of LPS is 50-1000. mu.g/ml.

According to an embodiment of the invention, the LPS concentration is 50. mu.g/ml, 100. mu.g/ml, 500. mu.g/ml, 1000. mu.g/ml.

Preferably, the LPS concentration is 500. mu.g/ml.

According to an embodiment of the invention, the dose of the coxsackie virus CVB3-A in the oncolytic medicament is 10⁹PFU。

Compared with the prior art, the invention has the beneficial effects that:

the invention provides the effect of LPS on coxsackie virus oncolytic, thereby expanding the application range of LPS, improving the coxsackie virus oncolytic effect, having the best effect at the concentration of 500 mu g/ml and enhancing the oncolytic effect by 20 percent.

Detailed Description

The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

Based on the angle of microorganisms, Coxsackie virus is enterovirus, LPS is a component of the outer wall of the cell wall of gram-negative bacteria and is glycolipid consisting of lipid and polysaccharide, and the synergistic oncolytic effect of the LPS provided by the invention on Coxsachievirus CVB3-A virus can increase the oncolytic efficacy by 20%.

To demonstrate the above synergistic effect, the examples of the present invention are as follows.

Example 1 establishment of a Virus oncolytic model

The viral cells of this example were derived from the cell line obtained in the present company's patent application No. 201410151863.9; vero cells were purchased from China Center for Type Culture Collection (CCTCC), Wuhan university.

1. Cell resuscitation

According to the principle of 'slow freezing and fast thawing', taking out cells to be thawed from a liquid nitrogen tank and a refrigerator at minus 80 ℃, quickly putting the cells into a water bath kettle preheated to 37 ℃ in advance, centrifuging at 1300rpm for 3min after complete thawing, discarding supernatant, gently resuspending by using 10% FBS complete culture medium, transferring the cells to 75cm from a freezing tube²Supplementing culture medium to 10ml, shaking, placing at 37 deg.C and 5% CO₂An incubator.

2. Cell exchange liquid

After 2-3 days of cell culture, the medium becomes acidic due to nutrient consumption and cell metabolite accumulation, and the phenol red indicator in the medium will turn yellow. At this time, if the cells do not satisfy the passage conditions, the cells need to be changed. When the culture medium is changed, the old culture medium is directly taken out and discarded, and an equivalent amount of fresh culture medium is added.

3. Cell passage

When the confluent rate of the cells is 80-90%, the cells need to be passaged. Firstly, taking out the old culture medium, discarding, adding a proper amount of PBS (phosphate buffer solution) for washing twice, then sucking and discarding, then adding a small amount of pancreatin, placing the cells at 37 ℃ after fully contacting the cells, and placing the cells at 5% CO₂Digesting for 3min, observing cell rounding and dropping from plate bottom, and tapping culture flask; finally adding a proper amount of complete culture medium to suspend the cells, blowing and beating for 5-6 times to suspend and fully disperse the cells into single cells, evenly dividing the cells into 2-3 cell culture bottles, supplementing the culture medium, gently shaking uniformly, placing at 37 ℃ and 5% CO₂Culturing under the condition.

4. Cell cryopreservation

When the cells grow to the logarithmic growth phase, the state is good, and the cells are not needed for experiments or cell strains need to be preserved temporarily, the cells can be frozen. Firstly, according to the cell passage operation stepThe cells were digested by the procedure and resuspended in the ready-prepared cell freezing medium to a final cell concentration of approximately 5X 10⁶－1×10⁷Blowing and beating for 5-6 times to make the cells suspended and fully dispersed into single cells, and subpackaging the cells in a cell freezing tube; finally, the cells are placed in a programmed cooling box, placed in a refrigerator at minus 80 ℃ for 6 to 8 hours, and then can be transferred into a liquid nitrogen tank or directly placed at minus 80 ℃ for storage. The cells can be frozen at-80 ℃ for a short time, and need to be placed in a liquid nitrogen tank for a long time.

5. Cell counting

When the cells need to be counted, the cells are firstly stopped to be digested, resuspended by using a proper amount of culture medium and fully mixed, then 20 mu L of cell suspension is taken out and slowly dropped into a cell counting groove of a cell counting plate from the edge of the counting plate, and the cells are counted by using a cell counting instrument. The cells can be counted separately in 2 different fields and finally averaged. After the cell counting plate is used, the cell counting plate can be washed clean by ultrapure water, placed at room temperature and naturally dried, and then used for multiple times.

6. Virus production and neoplasia

6.1 Virus production

1) Culturing Vero cells at 225cm²Cell culture flasks (10% FBSDMEM medium) to a confluency of about 90%;

2) the culture medium was replaced with serum-free DMEM medium, and CVB3 virus was inoculated at an MOI of 0.1.

3) The virus is infected for about 24h, the cells are sampled and observed to be completely diseased, and the culture is stopped.

4) And filtering the virus liquid through a combined filter to obtain a virus harvest liquid.

5) After filtration, the mixture was split into 500. mu.L/tube and stored at-80 ℃ for further use.

6.2 animal experiments

6.2.1 nude mice tumor formation experiment

1) Dividing 4-6-week-old female BALB/c nude mice into 8 cages, and feeding under appropriate environmental conditions of temperature, humidity, light, noise and the like to provide sufficient food and water, wherein the food and the water need to be sterilized due to the defect of nude mouse immunity;

2) expanding cells to be tumorigenic to a 150mm cell culture plate, and culturing the cells in a large quantity to obtain enough tumor cells for tumorigenic;

3) digesting tumor cells for forming tumor when the cells grow to the logarithmic phase, stopping digestion by using a complete culture medium, fully suspending the cells into a single cell suspension, and centrifuging at 1300rpm for 3min to collect the cells;

4) removing the culture medium, adding a proper amount of PBS (precooled to 4 ℃) to remove the FBS remained on the cell surface, and centrifuging at 1300rpm, 3min and 4 ℃;

5) discarding PBS, adding appropriate amount of serum-free culture medium (precooled to 4 deg.C) to resuspend cells, counting cells, supplementing serum-free culture medium according to cell number, and adjusting cell concentration to 10⁷-10⁸Each/ml, put on ice;

6) taking 100 mu L of cell suspension, placing the cell suspension in a disposable syringe, discharging air in a needle, wiping and disinfecting the back of a nude mouse by using 75% alcohol, and inoculating cells under the skin of the nude mouse;

7) observing every day, and treating the mouse when the mouse tumor grows to be required by an electric experiment;

8) regularly observing the state of the mouse and measuring the tumor size of the human body, wherein the tumor size is calculated by the formula:

V(volume)＝[L×(W)²]/2。

example 2 in vitro oncolytic experiment of Coxsackie virus CVB3 after incubation with LPS

In this example, 10 Coxsackie virus CVB3 was mixed with LPS⁸PFU of Coxsackie virus CVB3 was incubated with the same concentration of LPS, 10 in the control group⁸PFU of Coxsackie virus CVB3, and infected tumor cells as a virus, respectively.

1. In vitro anti-tumor experiment of virus

1) Mixing A549, H460, MCF-7, SUN-398, etc. at a ratio of 5 × 10⁵Inoculating each well into a 12-well plate to be cultured until a cell monolayer is formed;

2) respectively infecting Coxsachieverus CVB3-A of an experimental group and a control group, wherein the infection amount is MOI (multiplicity of infection) of 0.1-1000;

3) the tumor cell lesions were observed under a microscope day by day, and the survival rate was counted for 72 hours in virus-infected culture, and the results are shown in Table 1.

Note: "-" indicates no cytopathic effect; "+" indicates 15% of the cells had a pathological effect; "+++" indicates that 50% -75% of the cells had a pathological effect; "+ +++" indicates that over 75% of the cells had a pathological effect.

As can be seen from Table 1, the killing effect of the coxsackie virus CVB3-A mixed bred by LPS on lung cancer cell strains A549 and H460, breast cancer cell strain MCF-7 and liver cancer cell strain SUN398 is enhanced.

Effect of LPS concentration on the oncolytic Effect of Coxsachievevirus CVB3-A

Cell line: a549 cells were purchased from the shanghai cell bank of chinese academy of sciences;

experimental animals: BALB/c nude mice 3 weeks old, body weight 15 + -3 g.

1) A549 cells in logarithmic growth phase were collected, washed 2 times with Phosphate Buffered Saline (PBS), and inoculated subcutaneously into the right axilla of BALB/c nude mice (1X 10)⁷One cell/one), tumor-bearing mice were obtained;

2) when the tumor grows to 7-8 mm³At this time, tumor-bearing mice were randomly divided into 5 experimental groups.

3) For experimental groups 1-5, LPS was injected intratumorally at 0, 50. mu.g/ml, 100. mu.g/ml, 500. mu.g/ml, 1000. mu.g/ml and the same dose of 10⁹Coxsachievirus CVB3-A from PFU (plaque forming Unit). The injection time is once every other day, the change of the tumor is observed on different days, and the size of the tumor is measured.

As can be seen from Table 1, the tumor volumes of the Coxsachievirus CVB3-A virus used with LPS at different concentrations were all smaller than that of the test group 1 (without LPS), and furthermore, the tumor volumes tended to decrease when the LPS was 50. mu.g/ml, 100. mu.g/ml, 500. mu.g/ml and 1000. mu.g/ml, respectively, with the most significant effect when the LPS was 500. mu.g/ml. It can be seen that for cost and efficiency reasons, the oncolytic potency of Coxsachievirus CVB3-A is the best and can be enhanced by 20% when the LPS is used at 500. mu.g/ml.

The invention is not limited solely to that described in the specification and the embodiments, and additional advantages and modifications will readily occur to those skilled in the art, and it is not intended to be limited to the specific details, representative embodiments and illustrative examples set forth herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.