CN111321170A

CN111321170A - Mbp gene overexpression type microglia as well as preparation method and application thereof

Info

Publication number: CN111321170A
Application number: CN202010173815.5A
Authority: CN
Inventors: 杨丽娟; 周美玲; 王姝蕾; 王芳; 肖继坪; 陈波; 易子寒; 华海蓉
Original assignee: Kunming Medical University
Current assignee: Kunming Medical University
Priority date: 2020-03-13
Filing date: 2020-03-13
Publication date: 2020-06-23

Abstract

The invention discloses an Mbp gene overexpression microglia as well as a preparation method and application thereof, and relates to the technical field of genetic engineering. The preparation method disclosed by the invention comprises the steps of inserting the Mbp gene into an over-expression lentivirus vector to obtain a recombinant lentivirus vector, then co-transfecting a host cell with the recombinant lentivirus vector and a lentivirus packaging plasmid to prepare the recombinant lentivirus with the Mbp gene over-expressed, and infecting microglia to obtain the Mbp gene over-expression microglia. The preparation method can prepare the Mbp gene overexpression type microglia, the microglia can stably and efficiently express the Mbp gene, the Mbp gene can play a normal biological role in the microglia, and reliable cell materials are provided for researching the specific functions, regulation and control mechanisms and the like of the Mbp in the microglia.

Description

Mbp gene overexpression type microglia as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an Mbp gene overexpression microglia as well as a preparation method and application thereof.

Background

Neuroinflammation refers to an inflammatory reaction that occurs in the central and peripheral nervous systems. Numerous studies have found that the occurrence and development of various central nervous diseases are closely related to neuroinflammation, such as Alzheimer's Disease (AD), parkinson's disease (PA20001809rkinson disease, PD), and cerebral infarction. Neuroinflammation causes neuronal degenerative necrosis, mainly through the production and release of inflammatory mediators, inflammatory cytokines and oxidative stress products, which trigger brain damage. Bad prognosis of diseases related to neuroinflammation seriously affects the life quality of patients, huge treatment cost brings heavy burden to families and society, and researches show that PD brings about 170 billion yuan of economic burden to China every year. But also its poor prognosis seriously affects the quality of life for patients and families. Therefore, the deep research on the generation mechanism of the neuroinflammation is expected to provide a new thought and theoretical basis for the prevention and treatment of central nervous system diseases.

Activated microglia play an important role in the development and development of diseases related to neuroinflammation. Microglia evolve from monocytes in the blood. Generally, microglia in the central nervous system are in a quiescent state, and pathological conditions such as central nervous system diseases, stroke, etc. can lead to the activation of microglia. Activated microglia cause inflammatory damage to the central nervous system and nerve cell death by secreting inflammatory mediators, co-activating astrocytes, causing release of a large number of free radicals, nitric oxide, proteases, inflammatory cytokines, and the like. In addition, activated microglia and the cell inflammation factors generated by the activated microglia can also damage the blood brain barrier structure, so that T lymphocytes, monocytes, neutrophils and the like in peripheral blood can easily enter the brain, and can generate more inflammation mediators together with astrocytes and the like, thereby further causing inflammation cascade reaction and aggravating the damage of brain tissues.

MBPs are important constituents of myelin proteins and are distributed mainly in myelinating cells of the central and peripheral nervous system (oligodendrocytes and Schwann cells). MBPs are expressed in macrophages, and research by Ling EA et al shows that the Mbp gene is expressed in two MCs. Meanwhile, the research group found that the expression of MBPs in activated microglia cells was increased. However, the specific function and regulation mechanism of MBPs in microglia is still unclear and has not been reported. The research on the specific functions and regulation mechanisms of MBPs in microglia lacks corresponding cell materials.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an Mbp gene overexpression microglia as well as a preparation method and application thereof. The preparation method provided by the invention can be used for preparing the Mbp gene overexpression type microglia, the microglia can stably express the Mbp gene, the gene can play a normal biological function in the microglia, and the invention provides reliable cell materials for researching the specific function, regulation mechanism and the like of the Mbp in the microglia.

The invention is realized by the following steps:

in one aspect, the present invention provides a method for preparing Mbp gene-overexpressing microglia, which comprises the steps of:

recombinant overexpression lentivirus packaging step: inserting the Mbp gene into an overexpression lentiviral vector to obtain a recombinant lentiviral vector, and then co-transfecting a host cell with the recombinant lentiviral vector and a lentiviral packaging plasmid to prepare the Mbp gene overexpression recombinant lentivirus;

wherein the overexpression lentiviral vector is LentiORF pLEX-MCS;

a cell infection step: infecting microglia with the Mbp-overexpressed recombinant lentivirus to obtain Mbp gene-overexpressed microglia.

The preparation method provided by the invention selects a specific overexpression lentiviral overexpression vector LentiORFpLEX-MCS to prepare the recombinant lentivirus, and then the lentivirus is used for infecting microglia, so that the Mbp gene can be stably expressed in the microglia and can play a normal biological function, and the microglia has a corresponding phenotype or characteristic; can be used for researching the specific function and regulation mechanism of MBP in microglia and the like, and provides reliable cell materials for the MBP.

Alternatively, in some embodiments of the invention, the packaging plasmid comprises pVSV-G, pRev and pRRE.

Alternatively, in some embodiments of the invention, the host cell is a 293T cell.

It should be noted that the host cell may be selected according to the actual situation, including but not limited to 293T cell, and may be other types of cells, which are within the scope of the present invention.

Alternatively, in some embodiments of the invention, the species source of microglia is a mammal.

Alternatively, in some embodiments of the invention, the mammal is selected from any one of mouse, rat, pig, cow, horse and sheep.

It should be noted that, according to the research needs, the preparation method of the present invention can select suitable species-derived microglia as target cells, including but not limited to mouse, rat, pig, cow, horse and sheep, and other mammalian microglia can also be used as target cells for cell infection, and it is within the scope of the present invention to be considered as any mammalian microglia.

Alternatively, in some embodiments of the invention, the mammal is a mouse.

Alternatively, in some embodiments of the invention, the Mbp protein encoded by the Mbp gene has the amino acid sequence shown in SEQ ID No. 1:

MASQKRPSQRSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFSGDRGAPKRGSGKVPWLKQSRSPLPSHARSRPGLCHMYKDSHTRTTHYGSLPQKSQHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQKPGFGYGGRASDYKSAHKGFKGAYDAQGTLSKIFKLGGRDSRSGSPMARR。

it should be noted that, one skilled in the art can easily perform the operation of substitution or deletion or addition of one or more amino acids based on SEQ ID No.1 and make the resulting MBP protein variant have the same biological function as the MBP protein of SEQ ID No.1, and based on this, the MBP protein of the present invention may also be the MBP protein variant having the equivalent biological function obtained by the operation of substitution or deletion or addition of one or more amino acids based on SEQ ID No. 1.

Alternatively, in some embodiments of the invention, the nucleotide sequence of the Mbp gene is set forth in SEQ ID No.2 (CDS):

atggcatcacagaagagaccctcacagcgatccaagtacctggccacagcaagtaccatggaccatgccaggcatggcttcctcccaaggcacagagacacgggcatccttgactccatcgggcgcttctttagcggtgacaggggtgcgcccaagcggggctctggcaaggtaccctggctaaagcagagccggagccctctgccctctcatgcccgcagccgtcctgggctgtgccacatgtacaaggactcacacacgagaactacccattatggctccctgccccagaagtcgcagcacggccggacccaagatgaaaacccagtagtccatttcttcaagaacattgtgacacctcgaacaccacctccatcccaagggaaggggagaggcctgtccctcagcagatttagctggggggccgaggggcagaagccaggatttggctacggaggcagagcttccgactataaatcggctcacaagggattcaagggggcctacgacgcccagggcacgctttccaaaatctttaagctgggaggaagagacagccgctctggatctcccatggcgagacgctga。

the Mbp gene transcripts, 17, were selected to construct the CDS region (SEQ ID No.2) of transcript 1, myolin basic protein isofomm 1, into an overexpressed lentiviral vector, creating a recombinant lentiviral vector with Mbp gene overexpression, because the transcripts encoded the longest Mbp isoform, with a molecular weight of about 21.5kDa, predominantly localized to the nucleus, capable of interacting with key molecules of the nuclear signaling pathway and performing a specific biological function.

The nucleotide sequence of the Mbp gene may be codon-optimized according to the actual circumstances, and the sequence of the Mbp gene of the present invention may be a nucleotide sequence that has been variously optimized, so long as it encodes the Mbp protein described above, and it is within the scope of the present invention.

In another aspect, the present invention provides an Mbp gene-overexpressing microglia produced by the method of any one of the above.

The Mbp gene overexpression type microglia provided by the invention can stably express the MBP protein, has the corresponding characteristic of MBP protein overexpression, and is an ideal cell material for researching the specific function, regulation mechanism and the like of the MBP in the microglia.

In another aspect, the present invention provides the use of the recombinant microglia as described above in the study of Mbp gene function or its regulatory mechanism, or in the screening of drugs for treating neuroinflammation.

It should be noted that the application provided by the present invention is not limited to the fields of Mbp gene function and regulation mechanism research, drug screening, etc. the recombinant microglia provided by the present invention can be used in other fields, which also belongs to the protection scope of the present invention.

Alternatively, in some embodiments of the invention, the study is for the purpose of diagnosis or treatment of a non-disease.

In another aspect, the present invention provides a vector composition for preparing Mbp gene overexpressed microglia, which comprises a packaging plasmid and a recombinant lentiviral vector into which an Mbp gene is inserted, wherein the packaging plasmid is used for transfecting a host cell in cooperation with the recombinant lentiviral vector to prepare a recombinant lentiviral vector in which the Mbp gene is overexpressed;

the framework of the recombinant lentiviral vector is LentiORF pLEX-MCS;

the recombinant lentivirus is used to transfect microglia to obtain Mbp gene over-expressed microglia.

The vector composition can be used for preparing the Mbp gene overexpression microglia. The microglia have a corresponding phenotype or characteristic; can be used for researching the specific function and regulation mechanism of MBP in microglia and the like, and provides reliable cell materials for the MBP.

Alternatively, in some embodiments of the invention, the mammal is a mouse

Alternatively, in some embodiments of the invention, the amino acid sequence of the Mbp protein encoded by the Mbp gene is set forth in SEQ ID No. 1.

Alternatively, in some embodiments of the invention, the nucleotide sequence of the Mbp gene is set forth in SEQ ID No. 2.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a plasmid map of lentivirus vector LentiORF pLEX-MCS.

FIG. 2 shows the results of the sequencing in example 1.

FIG. 3 is a plasmid map of the packaging plasmid pVSV-G.

FIG. 4 is a plasmid map of the packaging plasmid pRev.

FIG. 5 is a plasmid map of packaging plasmid pRRE.

FIG. 6 shows the results of measuring the expression level of Mbp mRNA of Mbp gene-overexpressing microglia prepared in example 1 in experiment 1.

FIG. 7 shows the results of Western blot detection of MBP protein expression levels in Mbp gene-overexpressed microglia prepared in example 1 in Experimental example 1.

Fig. 8 is a result of measuring the cell morphology of Mbp gene-overexpressing microglia prepared in example 1 in experimental example 2, in which a: morphology of cells of the over-expression control group (i.e. cells of the empty vector control group (pLEX-NC)), b: mbp gene-overexpressing microglia prepared in example 1.

Fig. 9 is a graph showing the results of examining the cell migration ability of Mbp gene-overexpressed microglia prepared in example 1 in experimental example 2, wherein a: morphology of cells of the over-expression control group (i.e. cells of the empty vector control group (pLEX-NC)), b: mbp gene-overexpressing microglia prepared in example 1.

FIG. 10 is a plasmid map of pLV6 lentiviral overexpression vector of comparative example 1.

FIG. 11 is a comparison of the expression levels of Mbp mRNA in microglia overexpressing two Mbp genes in comparative example 1.

FIG. 12 is a comparison of MBP protein expression levels of two Mbp gene overexpressed microglia in comparative example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The present invention provides a method for preparing Mbp gene overexpressed microglia, which comprises the following steps:

1 construction of recombinant lentiviral vectors containing Mbp Gene

(1) Based on the sequence of the Mbp Gene (Gene ID:17196) of mice, the primers shown in Table 1 below were designed, with NotI and BamHI and protective bases added to the upstream and downstream primers, respectively:

TABLE 1

The primers were synthesized by Suzhou Jima Gen GmbH, dissolving each primer to a concentration of 50. mu.M, adding the same volume of each primer to the same 1.5ml centrifuge tube, and mixing to obtain primer mix.

(2) The first round of PCR reaction was performed by the following PCR system:

primer mix (50. mu.M)	6μl
		10×Pfu Buffer(+Mg²⁺)	5μl
dNTP(0.2mM)	1μl
		Y3858-1(50μM)	1μl
Y3858-14(50μM)	1μl
		ddH₂O	36μl
Pfu DNA polymerase(100U)	0.3μl

Description of the drawings: in the table, the primer mix is a mixture of 14 different primers from Y3858-1 to Y3858-14, which are used as templates for the first round of PCR reaction, and Y3858-1 and Y3858-14 are respectively upstream and downstream primers for the PCR reaction; pfu DNApolymerase was purchased from petunia bio, cat #: D7216.

the cycling conditions were as follows:

(3) after the first round of PCR is finished, carrying out a second round of PCR reaction, wherein a template is a product of the first round of PCR reaction, and a PCR system comprises the following components:

description of the drawings: the second round of PCR amplification conditions are the same as the first round, but the template used is the amplification product of the first round of PCR, and the reaction conditions are the same as the first round. The first round of PCR can obtain a mixture of non-single-band PCR products mixed with the target gene band, and then the first round of PCR products is used as a template, and the second round of PCR can obtain a single target gene band.

(4) After completion of the PCR reaction, the gene fragment was recovered by Agarose electrophoresis and cutting using a DNA gel recovery kit (Tiangen Biochemical technology Co., Ltd., product No. DP 209-03).

(5) The Mbp gene fragment was digested with NotI and BamHI at 37 ℃ for 2 hours in the following manner:

10×Buffer	5μl
		mmu-Mbp	15μl
NotI	1μl
		BamHI	1μl
ddH₂O	28μl

the vector LentiORF pLEX-MCS (hereinafter abbreviated as pLEX, the structure of which is shown in FIG. 1) was digested with NotI and BamHI at 37 ℃ for 2 hours in the following manner:

10×Buffer	5μl
		pLEX	5μl
NotI	1μl
		BamHI	1μl
ddH₂O	38μl

after the enzyme digestion, electrophoresis is carried out, and the Mbp gene fragment and the vector pLEX are recovered by using a DNA gel recovery kit. The double-digested Mbp gene fragment (nucleotide sequence shown in SEQ ID NO.2) and the linearized vector were ligated by T4 DNaligase (Thermo Scientific, cat # EL0011) at 22 ℃ for 2 hours in the following system:

(6) preparation of competent cells:

a single colony was picked from a fresh plate cultured at 37 ℃ for 16 hours, transferred to a 1L flask containing 100ml of LB medium, and cultured with vigorous shaking at 37 ℃ for 3 hours (rotary shaker, 300 rpm).

The bacteria were transferred aseptically to a sterile, single-use, ice-pre-cooled 50ml polypropylene tube, placed on ice for 10 minutes and the culture cooled to 0 ℃.

The cells were recovered by centrifugation at 4000 rpm for 10 minutes at 4 ℃. The culture was poured out and the tube was inverted for 1 minute to drain the last traces of culture.

With 10ml of 0.1mol/L CaCl precooled with ice₂Resuspend each pellet and place on an ice bath.

Every 50ml of initial culture was 2ml of ice-pre-cooled 0.1mol/L CaCl₂Resuspend each cell pellet (containing 20% glycerol). Cells were divided into aliquots (100. mu.l/piece) and stored frozen at-70 ℃.

(7) Transformation of the ligation products into competent cells

The competent cells were removed from-70 ℃, placed on ice for 4 minutes in the centrifuge tube containing the competent cells, after thawing the competent cells, 10 μ l of ligation product was added, the contents gently mixed and placed on ice for 30 minutes.

The centrifuge tube was placed on a test tube rack placed in a water bath preheated to 42 ℃ for 90 seconds without shaking the centrifuge tube.

The centrifuge tubes were quickly transferred to an ice bath and the cells were allowed to cool for 3 minutes.

To each tube was added 800. mu.l LB medium (without antibiotics) and the tubes were then transferred to a 37 ℃ shaker at 250 rpm for 45 minutes to resuscitate the bacteria.

200. mu.l of the cultured cells were applied evenly to LB plates containing 50. mu.g/ml Ampicillin.

After the liquid on the plate was absorbed, the plate was placed upside down in an incubator at 37 ℃ and incubated for 16 hours.

Clone colonies were picked from the plates, plasmids were mini-extracted and positive clones were picked for identification.

4 individual, filled colonies were picked from the cultured plate, placed in a test tube containing 5ml (50. mu.g/ml Kanamycin) of LB medium, and cultured in a shaker at 37 ℃ for 16 hours at 250 rpm.

And extracting plasmids from the cultured bacterial liquid by using a plasmid miniprep kit (Tiangen Biochemical, DP104-02) (the plasmid extraction steps are detailed in the specification of the plasmid miniprep kit).

Carrying out double enzyme digestion identification on the extracted plasmid, wherein each tube of reaction system comprises the following steps:

10×Buffer	1μl
		plasmids	1μl
NotI	0.5μl
		BamHI	0.5μl
ddH₂O	7μl

Carrying out enzyme digestion at 37 ℃ for 1 hour, carrying out electrophoresis, and obtaining a clone corresponding to the band obtained by enzyme digestion in a region corresponding to the size of the target band, namely a positive clone.

(8) Sequencing and verifying recombinant plasmids and extracting in large quantity:

200 mul of bacterial liquid corresponding to the positive clone was sequenced and the remaining bacterial liquid was preserved with glycerol.

And (3) comparing a sequencing result (shown in figure 2) with a target gene sequence (SEQ ID NO.1), and after the sequencing result is identified to be correct, inoculating a strain LB culture medium with preserved glycerol bacterial liquid, and performing a large amount of plasmid extraction to obtain a sufficient amount of recombinant plasmids to obtain an over-expression lentivirus vector LentiORF pLEX-MCS (namely a recombinant lentivirus vector) containing an Mbp gene fragment.

2 viral packaging

HEK 293T cells were cultured in 10cm dishes with 10% FBS in high glucose DMEM until 80-90% confluency, and 15cm dishes were plated. Placing in a cell culture box at 37 deg.C and 5% CO₂The culture was carried out overnight. The over-expressed lentiviral vector containing the Mbp gene fragment was co-transfected with the packaging plasmids pVSV-G (see FIG. 3), pRev (see FIG. 4) and pRRE (see FIG. 5) into 293T cells according to the Lipofectamine TM 2000 protocol, the medium was harvested after 72h of transfection, the over-expressed lentiviral stock containing the Mbp gene was obtained by filtration through a 0.45 μm filter, labeled and stored at-80 ℃.

3 cell infection

(1) Taking mouse microglia (BV2) in logarithmic growth phase, and processing according to 2.5 × 10⁵The concentration of each cell/well was inoculated into a 24-well plate, 500. mu.L of complete medium was added thereto at 37 ℃ and 5%CO₂Culturing in an incubator overnight; the next day, preparing virus diluent (DMEM medium 400 μ L + 5 μ g/mL Polybrene final concentration), adding lentivirus stock solution into the diluent at a ratio of 1:9, preparing three virus solutions with different concentrations, adding into corresponding culture wells of each group, and simultaneously establishing control (blank, negative) at 37 deg.C and 5% CO₂The incubator was incubated overnight. The virus solution after cell infection is removed in 12-24h, 500 mu L of complete culture solution is added, the culture is continued for 72-96h at 37 ℃ and 5% CO2 incubator, the total RNA and total protein of the cells are extracted, and the overexpression effect of the Mbp gene is detected by a real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) or immunoblotting (western blot) method, and the result is shown in figure 6 and figure 7.

(2) Screening of MBP gene overexpression stable transgenic cell strain

Microglia in logarithmic growth phase were plated in 96-well plates (2 × 10)⁵One/well), adding puromycin for screening 7d after adherence, setting 6 parallel wells for each concentration, determining screening concentration, observing for one week, and finally determining to screen stable transformants by puromycin with the concentration of 2 ug/mL. And (3) under the pressure of the determined puromycin concentration, adding puromycin for screening for two weeks, then carrying out amplification culture, and screening stable transformants from the microglia in the step (1) to obtain the stable Mbp gene overexpression type microglia.

Experimental example 1

Verifying the overexpression effect of the Mbp gene overexpression microglia.

RNA and protein samples were extracted from the partially Mbp gene-overexpressing microglia cells obtained in example 1, and the effect of Mbp gene overexpression was verified.

(1) The Real-time fluorescent quantitative PCR (Real-time RT-PCR) is used for detecting the mRNA expression level of the Mbp gene, 0.25 percent of pancreatin is digested and cells are collected, Total RNA is extracted by a Trizol reagent, cDNA is synthesized by reverse transcription according to the specification of a reverse transcription kit, the Total reaction system is 20uL, 1uL of cDNA is used as an upstream primer and a downstream primer (final concentration is 300nM) respectively, 7uL of DEPC water, and 2 × Mix SYBR Green I fluorescent reaction solution ROX 10 uL. reaction conditions are that the reaction conditions are pre-denatured at 95 ℃ for 2min → denaturation at 95 ℃ for 30s in a cycle and annealing at 60 ℃ for 30s, 40 cycles are set, fluorescent signals are photographed and collected at the end of extension of each cycle, and an amplification curve is drawn.

After 40 cycles, setting the reaction steps of 95 ℃ 15s → 60 ℃ 30s → 95 ℃ 15s, photographing in the temperature rise process from 60 ℃ to 95 ℃ to collect the whole fluorescence signal, and drawing a dissolution curve. By use of 2^-△△CtThe assay determines Mbp mRNA expression, setting 3 duplicate wells per sample. The results are shown in FIG. 6, and the experimental results show that the expression level of Mbp mRNA in Mbp gene-overexpressing microglia is increased by five thousand times compared with the control cells.

In FIG. 6, pL-NC is empty vector control cell; b: wild type cells, cells without any treatment, cells of the control group and the Mbp group are constructed by infecting different viruses on the basis of the cells; mbp gene-overexpressing microglia; 72H: representing the time of virus infection, at which point cellular RNA was extracted for corresponding assays.

In general, total RNA of cells is extracted 72 hours after the virus infects the cells to detect the mRNA expression level, and total protein of the cells is extracted 96 hours after the virus infects the cells to detect the protein expression level.

(2) Western blot is used for detecting the expression level of the Mbp protein, the result is shown in figure 7, and the experimental result shows that the expression level of the MBP protein of the Mbp gene overexpression microglia is obviously increased compared with the cells of a control group. In fig. 7: pLEX-NC is empty vector control cell; blank: wild type cells, cells without any treatment, cells of the control group and the Mbp group are constructed by infecting different viruses on the basis of the cells; mbp gene-overexpressing microglia; 96H: representing the time of viral infection at which the proteins of the cells were extracted for the corresponding assay.

Experimental example 2

Detection of cellular morphology of Mbp Gene-overexpressing microglia

(1) The method comprises the following steps: the morphological structure of the cells is observed by a phase contrast inverted microscope and photographed and recorded.

The results are shown in FIG. 8, which shows: compared with the cells of the control group, the cell morphology of the Mbp overexpression group is mainly branched cells.

(2) The method comprises the following steps: the tranwell assy experiment is used for detecting the migration capacity of the cells, and the specific operation is as follows:

taking cells in logarithmic growth phase, digesting and counting the cells, taking 40,000 cells, adding the cells into the upper layer of a transwell chamber, wherein the total volume is 100ul, no serum is contained, and 3 chambers are used in each group; the lower layer of the chamber was filled with 600ul of medium containing 10% serum at 37 ℃ with 5% CO₂After 24 hours of culture in the incubator, the chamber was fixed with 4% paraformaldehyde, and then the cells of the upper membrane were gently wiped off and the cells of the lower membrane were stained with DAPI. The stained cells of the lower membrane were photographed under a fluorescent microscope, and 5 fields of view, 20X objective, were randomly selected for each chamber. And counting the number of cells in each picture by using cell counting software, adding the number of 5 visual fields in each hole to form a data value, wherein each group of three multiple holes has three data values, and each group can be subjected to significance difference test by t-test.

The results are shown in FIG. 9, which shows: the number of cells that migrated by Mbp-overexpressing cells was significantly increased compared to control cells, suggesting that Mbp may promote cell migration.

Comparative example 1

This comparative example prepares Mbp gene-overexpressing microglia in substantially the same manner as in example 1, except that this comparative example uses pLV6 lentiviral overexpression vector (see FIG. 10) instead of LentiORF pLEX-MCS.

The expression levels of Mbp mRNA and Mbp protein were compared between Mbp gene-overexpressed microglia of comparative example 1 and Mbp gene-overexpressed microglia of example 1, and the results are shown in fig. 11 and 12.

As can be seen from the results in the figure, after the Mbp overexpression lentivirus constructed by using the pLV6 overexpression lentivirus vector infects cells, the intracellular Mbp mRNA expression level is up-regulated by 2.37 times (FIG. 11), but the Mbp protein expression level is not obviously changed (FIG. 12), which indicates that a high-efficiency and stable Mbp overexpression cell strain cannot be constructed by using the pLV6 lentivirus vector. The selection of the overexpression lentiviral vector LentiORF pLEX-MCS can construct a high-efficiency and stable Mbp overexpression cell strain, which is unexpected for the skilled person in the art.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> university of Kunming medical science

<120> Mbp gene overexpression type microglia cell and preparation method and application thereof

<160>2

<170>PatentIn version 3.5

<210>1

<211>195

<212>PRT

<213> Artificial sequence

<400>1

Met Ala Ser Gln Lys Arg Pro Ser Gln Arg Ser Lys Tyr Leu Ala Thr

1 5 10 15

Ala Ser Thr Met Asp His Ala Arg His Gly Phe Leu Pro Arg His Arg

20 25 30

Asp Thr Gly Ile Leu Asp Ser Ile Gly Arg Phe Phe Ser Gly Asp Arg

35 40 45

Gly Ala Pro Lys Arg Gly Ser Gly Lys Val Pro Trp Leu Lys Gln Ser

50 55 60

Arg Ser Pro Leu Pro Ser His Ala Arg Ser Arg Pro Gly Leu Cys His

65 70 75 80

Met Tyr Lys Asp Ser His Thr Arg Thr Thr His Tyr Gly Ser Leu Pro

85 90 95

Gln Lys Ser Gln His Gly Arg Thr Gln Asp Glu Asn Pro Val Val His

100 105 110

Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro Ser Gln Gly

115 120 125

Lys Gly Arg Gly Leu Ser Leu Ser Arg Phe Ser Trp Gly Ala Glu Gly

130 135 140

Gln Lys Pro Gly Phe Gly Tyr Gly Gly Arg Ala Ser Asp Tyr Lys Ser

145 150 155 160

Ala His Lys Gly Phe Lys Gly Ala Tyr Asp Ala Gln Gly Thr Leu Ser

165 170 175

Lys Ile Phe Lys Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser Pro Met

180 185 190

Ala Arg Arg

195

<210>2

<211>588

<212>DNA

<213> Artificial sequence

<400>2

atggcatcac agaagagacc ctcacagcga tccaagtacc tggccacagc aagtaccatg 60

gaccatgcca ggcatggctt cctcccaagg cacagagaca cgggcatcct tgactccatc 120

gggcgcttct ttagcggtga caggggtgcg cccaagcggg gctctggcaa ggtaccctgg 180

ctaaagcaga gccggagccc tctgccctct catgcccgca gccgtcctgg gctgtgccac 240

atgtacaagg actcacacac gagaactacc cattatggct ccctgcccca gaagtcgcag 300

cacggccgga cccaagatga aaacccagta gtccatttct tcaagaacat tgtgacacct 360

cgaacaccac ctccatccca agggaagggg agaggcctgt ccctcagcag atttagctgg 420

ggggccgagg ggcagaagcc aggatttggc tacggaggca gagcttccga ctataaatcg 480

gctcacaagg gattcaaggg ggcctacgac gcccagggca cgctttccaa aatctttaag 540

ctgggaggaa gagacagccg ctctggatct cccatggcga gacgctga 588

Claims

1. A method for preparing Mbp gene-overexpressing microglia, comprising the steps of:

wherein the overexpression lentiviral vector is LentiORF pLEX-MCS;

2. The method of claim 1, wherein the packaging plasmid comprises pVSV-G, pRev and pRRE.

3. The method of claim 1 or 2, wherein the host cell is a 293T cell.

4. The method of claim 1 or 2, wherein the species source of microglia is a mammal.

5. The method according to claim 4, wherein the mammal is selected from any one of mouse, rat, pig, cow, horse and sheep;

preferably, the mammal is a mouse.

6. The method according to claim 1 or 2, wherein the Mbp protein encoded by the Mbp gene has the amino acid sequence shown in SEQ ID No. 1;

preferably, the nucleotide sequence of the Mbp gene is shown in SEQ ID NO. 2.

7. An Mbp gene-overexpressing microglia cell produced by the method of any one of claims 1-6.

8. Use of the Mbp gene-overexpressing microglia according to claim 7 in the study of Mbp gene function or its regulatory mechanisms, or in the screening of drugs for the treatment of neuroinflammation.

9. A vector composition for producing Mbp-gene-overexpressing microglia, which comprises a packaging plasmid for transfecting a host cell in cooperation with a recombinant lentiviral vector into which an Mbp gene is inserted to produce a recombinant lentivirus in which an Mbp gene is overexpressed;

the framework of the recombinant lentiviral vector is LentiORF pLEX-MCS;

10. The vector composition of claim 9, wherein the packaging plasmid comprises pVSV-G, pRev and pRRE;

preferably, the host cell is a 293T cell;

preferably, the species source of microglia is a mammal;

preferably, the mammal is selected from any one of mouse, rat, pig, cow, horse and sheep;

preferably, the mammal is a mouse;

preferably, the amino acid sequence of the MBP protein coded by the Mbp gene is shown in SEQ ID NO. 1;

preferably, the nucleotide sequence of the Mbp gene is shown in SEQ ID NO. 2.