CN111955417A

CN111955417A - Method for constructing mouse pulmonary cytomegalovirus latent infection model

Info

Publication number: CN111955417A
Application number: CN202010875625.8A
Authority: CN
Inventors: 张志辉; 孙艺凝
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-08-19
Filing date: 2020-08-19
Publication date: 2020-11-20
Anticipated expiration: 2040-08-19
Also published as: CN111955417B

Abstract

The invention discloses a method for constructing a mouse pulmonary cytomegalovirus latent infection model, which comprises the steps of continuously feeding a drug prednisone to a mouse through an esophagus, injecting MCMV virus into the abdominal cavity of the mouse, and identifying the pulmonary cytomegalovirus latent infection state by using a molecular biology detection technology. The invention induces cytomegalovirus to generate latent infection and cytomegalovirus to generate latent infection in the lung of the mouse by a method of orally taking medicine prednisone and injecting MCMV virus in the abdominal cavity for the mouse, and can construct an animal model of the cytomegalovirus latent infection, and the mouse model shows the typical characteristics of the cytomegalovirus latent infection in the lung: the lung tissue presents virus expression related protein. The model can be applied to scientific research in the related field of cytomegalovirus infection, and lays an experimental animal foundation for exploring a pathogenic mechanism of cytomegalovirus infection.

Description

Method for constructing mouse pulmonary cytomegalovirus latent infection model

Technical Field

The invention relates to the technical field of bioengineering, and particularly relates to a method for constructing a mouse pulmonary cytomegalovirus latent infection model.

Background

The infection rate of Human Cytomegalovirus (HCMV) in people reaches 60-100%, wherein the HCMV infection rate in newborn babies in China reaches 96.2%, and the infection rate is related to factors such as sanitary conditions, economic conditions, geographical conditions and the like. HCMV is one of herpes viruses, has the characteristic of herpes virus infection, namely, once a human body is infected with the herpes virus, the herpes virus is carried for life, and can cause the bad fate of congenital nervous system dysplasia, induction of rejection of transplanted organs, weakening of immune defense function and the like, thereby seriously threatening life and health, but the current treatment and prevention measures are few.

Cytomegalovirus (CMV) has significant species specificity, and HCMV is not effective in infecting non-human experimental animals, especially rats. Therefore, many studies have been conducted to establish an animal model in which mice infected with Murine Cytomegalovirus (MCMV) simulate the in vivo infection process of HCMV. Currently, a mouse model of MCMV viral infection is constructed in the following manner: (1) inoculating MCMV through placenta in a microinjection mode to establish an MCMV intrauterine infection model; (2) establishing a BALB/c mouse MCMV hepatitis model by using a recombinant virus strain inserted with a LacZ gene; (3) mouse model of peripheral blood infection with MCMV was injected xenogeneically. In addition, moustache shadow and the like (2006) disclose that a BALB/c old mouse model for liver injury caused by reactivation of HCMV congenital latent infection is established, namely, old mice infected by HCMV virus are randomly activated by cyclophosphamide, and an HCMV latent reactivation infected mouse model is established and used for researching liver injury caused by reactivation of human HCMV latent infection to the middle-aged and old people (moustache shadow, Wangming. human cytomegalovirus congenital latent infection reactivation causes liver injury of old mice [ J ]. world China digestion journal, 2006, 14 (12): 1146-1150.); wangyu et al (2015) disclose the establishment of a mouse brain latent cytomegalovirus activation model, which comprises the steps of adopting BALB/c young mice, injecting Smith Strain MCMV 500 PFU/5 mu L into a right ventricle through the center of an equilateral triangle with a right lateral ear and an eye connecting line as a base, culturing for 16 weeks, and then injecting Lipopolysaccharide (LPS) into an abdominal cavity and a lateral ventricle according to a weight of 15 mu g/kg (approximate lethal dose) to establish a mouse brain latent cytomegalovirus activation model (Wangyu, Liujun, Liuying, and the like; the establishment of the mouse brain latent cytomegalovirus activation model [ J ] modern biomedical progress 2015 23 (4414): 4418.). However, no report related to the construction of an experimental mouse model of pulmonary latent infection MCMV is found at present, and due to the difference of organ tissues and the difference of detection substances, the existing method is not suitable for the establishment of pulmonary MCMV latent infection, and the existing method does not evaluate the MCMV latent infection state on a molecular level, and meanwhile, the construction time is too short, the evaluation means is few, and the influence of MCMV body tissues cannot be comprehensively evaluated. Therefore, in order to deeply research the pathogenic mechanism of human CMV, evaluate the antiviral treatment effect of the drug and research and develop vaccines, a mouse lung cytomegalovirus latent infection model needs to be established urgently.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a method for constructing a mouse pulmonary cytomegalovirus latent infection model.

The above object of the present invention is achieved by the following technical solutions:

a method for constructing a mouse pulmonary cytomegalovirus latent infection model comprises the steps of continuously feeding a drug prednisone to a mouse through an esophagus, injecting MCMV virus into the abdominal cavity of the mouse, and identifying the pulmonary cytomegalovirus latent infection state.

The invention firstly feeds the mice with the drug prednisone continuously through the esophagus, then injects MCMV virus into the abdominal cavity of the mice, and identifies the latent infection state of the pulmonary cytomegalovirus. The invention successfully constructs a lung CMV latent infection animal model, in particular to a mouse MCMV latent infection model which is highly homologous with human genome, good in economy and strong in reproductive capacity, and the experimental mouse model shows the typical characteristics of lung MCMV latent infection: the lung tissue presents virus expression related protein.

Preferably, the mice are continuously fed with the prednisone through esophagus, then the MCMV virus is injected into the abdominal cavity of the mice, and the latent infection state of the pulmonary cytomegalovirus is identified.

Preferably, the mice are continuously esophagogastrin for 7 days.

Preferably, the dose of the prednisone drug fed through the esophagus is 1 mg/kg.

Preferably, the MCMV virus is an MCMV Smith virus strain.

Preferably, the dose of the intraperitoneal injection of the MCMV virus is 5 x 10⁴PFU。

Preferably, the mouse is a female BALB/c mouse 6 to 8 weeks old.

Preferably, after injecting MCMV into the abdominal cavity to infect the mice, the mice are continuously raised for 16 weeks, and then the latent infection state of the MCMV in the lung is identified.

Preferably, the identification of the latent infection status of MCMV in the lung is performed by polymerase chain reaction, in vitro plaque assay and focused amplification assay to confirm the presence of latent infection of MCMV in the lung, indicating successful construction of latent MCMV infected mice when both MCMV RNA and 1VPA or FEA are negative in lung tissue, otherwise failing.

Further preferably, the method also comprises an in vitro plaque experiment and a focused amplification determination method MCMV.

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

the invention enables the lung of an experimental mouse to generate cytomegalovirus latent infection by continuously feeding prednisone through esophagus and injecting MCMV virus in the abdominal cavity, can construct an animal model of the pulmonary cytomegalovirus latent infection, and has novel construction method, but the construction method does not exist at present. Meanwhile, the model building method has long duration, and the evaluation mode is comprehensive, systematic and accurate, and is more accurate than the existing cytomegalovirus latent infection animal model.

Drawings

FIG. 1 is a flow chart of the construction of a mouse pulmonary cytomegalovirus latent infection model.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Evaluation techniques used in the following detailed description:

(1) PCR primer design

The following primers were used for all CMV-PCR and RT-PCR reactions. Primers were designed using DNASTAR software. The specificity of the mouse cytomegalovirus DNA polymerase (═ repair polymerase) sequences from genbank sequence PCR amplification products was confirmed by using an ABI-PRISM sequencer.

(2) Primer and method for producing the same

DNA pol：5’-GGGACCCTACTCCGACGACGTG-3’

DNA pol：3’-GCTCTGCTCTTCGATCGGTAGG-5’

(3)PCR

DNA was extracted from the tissue using QIAamp tissue kit (QIAGEN GmbH). DNA extracted from the homogenate was eluted in 100. mu.l distilled water and stored at-20 ℃ until analysis. In a total volume of 25. mu.l of PCR buffer (50mM KCl, 20mM TrisHCl [ pH8.4 ]]And 1.5mM MgCl₂) 200nM primer and 1.0U Taq DNA polymerase (GibcoBRL) were added to amplify the DNA. PCR was performed on a PerkinElmer 9700 thermocycler using the following procedure: initial denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 35 times 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 30s, and final extension at 72 ℃ for 7min, followed by holding at 4 ℃. The primers used to transcribe the DNA polymerase genes are listed above. Beta-actin transcripts served as cellular transcriptional controls. The amplification products were separated by electrophoresis on a 1% agarose gel and stained with ethidium bromide.

(4) RT-PCR and nested PCR

RNA was extracted from the tissue using Trizol reagent, dissolved in 100. mu.l of diethylpyrocarbonate water and stored at-80 ℃. RT reaction was performed for 20min with 1U of DNaseI at 37 ℃ to digest 1-5. mu.g of RNA. RT reactions were carried out in a total volume of 20. mu.l, containing 60mM KCl, 15mM Tris-HCl (pH8.4), 3mM MgCl₂10mM dithiothreitol, 20% (vol/vol) glycerol, 1mM dNTP triphosphate per primer, 12.5pM and 2.5U of the super-transcriptase. Primer annealing was performed at 25 ℃ for 10min, RT at 42 ℃ for 30min, followed by digestion with 1URNAseH, and denaturation at 95 ℃ for 5 min. To control DNA contamination, each sample was run in parallel without RT reaction.

(5) In Vitro Plaque Assay (IVPA)

For IVPA, mouse embryonic fibroblasts were cultured in Dulbecco's modified Eagle medium in 6-well plates. After centrifugation (1000g, 10min)5mL of homogenized tissue, 1mL of supernatant was placed in each well. The plates were then centrifuged 1000g, incubated at 37 ℃ in 5% CO2 for 3-4h, washed 3 times with PBS, and then covered with 3mL of 1% agar in DMEM. After 6-7 days of incubation (37C, 5% CO2), plates were fixed in 10% formalin, stained with 1% crystal violet, and analyzed for plaque formation using a low contrast microscope.

(6) Focused amplification assay (FEA)

Tissues were subjected to Dounce homogenization at 4 ℃ and homogenized for appropriate dilution in DMEM for infection with MEFs. Monolayers of MEFs (75 cm)²5X 10 in flask⁶Individual cells) were inoculated with 2mL of freeze-thawed and homogenized tissue and centrifuged (1000g, 30min at 20 ℃). After centrifugation, the supernatant was discarded, and the cells were washed twice with PBS. Virus replication was allowed to proceed at 37 ℃ for 72 hours in a 5% CO2 humidified atmosphere. After incubation, the indicators MEFs (PBS containing 0.05% wt/vol pancreatin and 0.02% wt/vol EDTA [ pH7.2 ] were detached with trypsin]) DMEM with 10% fetal calf serum was added. Cells were pelleted at 500g for 10min, taken up in PBS, and dissolved in extraction buffer of TRIzol reagent (GibcoBRL). RNA was isolated and viral RNA was analyzed by RT-PCR.

Example 1 establishment and evaluation of mouse pulmonary cytomegalovirus latent infection model

1. Processing for establishing mouse lung cytomegalovirus latent infection model

(1) Animal selection and treatment: female BALB/c mice 6 to 8 weeks old were used. After obtaining animal ethical review, mice were housed in an animal biosafety laboratory, level 2 laboratory. Mice were euthanized using a cervical dislocation method after inhalation of the anesthetic isoflurane. The mouse lungs and salivary glands were aseptically dissected, and the removed lungs and salivary glands were immediately frozen in liquid nitrogen and then stored in a-80 ℃ refrigerator.

(2) Esophagus feeding medicine: female BALB/c mice 6-8 weeks old were selected and fed with prednisone 1mg/kg for 7 days via esophageal feeding, oral prednisone being a glucocorticoid.

2. Construction of Lung latent MCMV infected mice

A purified MCMV Smith strain (VR-194/1981) was obtained from the American type culture Collection. By intraperitoneal injection of 5X 10⁴PFU MCMV Smith virus strains achieved primary MCMV infection. Mice were maintained for 16 weeks following MCMV infection. Respectively at the 2 nd,4. Mice were sacrificed at 8, 12, 16, 20, 24 weeks and mouse lung tissue was subsequently obtained.

3. Identification of latent infection status of pulmonary cytomegalovirus

The presence of MCMV latent infection was confirmed by Reverse transcription polymerase chain reaction (RT-PCR), In Vitro Plaque Assay (IVPA) and Focused amplification assay (FEA) methods. Obtaining mouse lung tissue and processing the lung tissue into lung tissue homogenate, wherein when MCMV RNA and IVPA or FEA in the lung tissue are negative, the successful construction of latent MCMV infected mice is indicated, otherwise, the construction fails. The results are shown in table 1, after the 16 th week of MCMV infection in mice, both MCMV RNA and IVPA or FEA were negative in lung tissue, confirming the presence of MCMV latent infection, indicating successful construction of latent MCMV infected mice.

TABLE 1 pulmonary tissue RNA, IVPA/FEA, DNA of mice at weeks 2, 4, 8, 12, 16, 20, 24

(5 mice per group; +: represents 1 mouse positive; -: represents 1 mouse negative)

The above description is of the preferred embodiment of the invention. It is to be understood that the invention is not limited to the particular embodiments described above, in that devices and structures not described in detail are understood to be implemented in a manner common in the art; those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or modify equivalent embodiments, without affecting the spirit of the invention, using the methods and techniques disclosed above, without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.

Claims

1. A method for constructing a mouse pulmonary cytomegalovirus latent infection model is characterized in that firstly, a mouse is continuously fed with prednisone through esophagus, then MCMV virus is injected into the abdominal cavity of the mouse, and the pulmonary cytomegalovirus latent infection state is identified.

2. The method of claim 1, wherein the mice are continuously esophagogastrin for 7 days.

3. The method of claim 2, wherein the dose of prednisone is 1 mg/kg.

4. The method of constructing according to claim 1, wherein the MCMV virus is an MCMV Smith virus strain.

5. The method of claim 4, wherein the intraperitoneal injection of the MCMV is performed at a dose of 5 x 10⁴PFU。

6. The method of claim 1, wherein the mouse is a female BALB/c mouse 6 to 8 weeks old.

7. The method of claim 1, wherein the incubation of the mice infected with MCMV is continued for 16 weeks after intraperitoneal injection, and then the latent infection status of MCMV in the lung is determined.

8. The method of claim 1, wherein the identification of the latent infection status of MCMV in lung is performed by polymerase chain reaction, in vitro plaque assay and focused amplification assay to confirm the presence of latent infection of MCMV in lung, wherein when MCMV RNA and IVPA or FEA are negative in lung tissue, successful construction of latent MCMV infected mouse is indicated, otherwise failure is indicated.

9. The method of claim 1, further comprising in vitro plaque assay and focused amplification assay (MCMV).