CN105420196A

CN105420196A - Construction method and application for stably expressing HPV16 E5 protein cell strain

Info

Publication number: CN105420196A
Application number: CN201510974888.3A
Authority: CN
Inventors: 钟儒刚; 李凡; 李劲涛; 汪杨俊琦
Original assignee: Beijing University of Technology
Current assignee: Beijing University of Technology
Priority date: 2015-12-23
Filing date: 2015-12-23
Publication date: 2016-03-23

Abstract

The invention relates to a construction method and application for stably expressing an HPV16 E5 protein cell strain. According to the construction method and application, an HPV16 E5 gene which is mediated by slow virus and provided with an His label is integrated into a Balb/c3T3 cell, and stable expression of the HPV16 E5 cancer gene is achieved. The cell strain can provide a stable cell platform for research of HPV 16 E5 protein biological characteristics and a pathogenic mechanism, and meanwhile a foundation is laid for prevention and control work of HPV-related tumors.

Description

The construction process of a kind of stably express HPV16 E5 albuminous cell strain and application

Technical field

The invention belongs to biomedicine field, be specifically related to construction process and the application of the strain of a kind of stably express HPV16E5 albuminous cell.

Background technology

Slow virus (Lentivirus) carrier is the gene therapy vector grown up based on I type human immunodeficiency virus.Distinguish general retroviral vector, foreign gene can be incorporated on host chromosome by lentiviral vectors effectively, thus reaches persistence expression.Lentiviral contains the genetic information required for packaging, transfection, stable integration, needs to utilize expression vector and packaging plasmid cotransfection cell simultaneously, carries out the packaging of virus in cell.Packaged virion is secreted in extracellular substratum, after centrifuging and taking obtains supernatant liquor, produces the virion of high titre, can be directly used in the infection of host cell.After goal gene enters into host cell, through reverse transcription, be incorporated into genome, thus high-caliber expression goal gene.In the experimental implementation that cell is relevant, being difficult to transfection according to a conventional method for some even cannot the cell of transfection, greatly can be improved the transduction efficiency of gene, with the high expression of the gene that achieves the goal by the experiment of lentivirus mediated.

Human papillomavirus (humanpapillomavirus, HPV) is the DNA tumour virus that a class can cause skin and mucous membrane tesselated epithelium propagation.The generation of the Several Kinds of Malignancies such as the infection of high-risk HPV 16 and cervical cancer, the esophageal carcinoma, laryngocarcinoma, nasopharyngeal carcinoma, mammary cancer develops closely related.HPVE5 oncogene causes and one of the key gene maintaining tumor development, and HPVE5 cancer protein of its coding is a kind of embrane-associated protein with high hydrophobicity, is mainly positioned in the structures such as cytolemma, endoplasmic reticulum, golgi body.Infect at HPV the commitment that related neoplasms formed, containing abundant E5ORF and E5 albumen in cell, and late in tumour cell, the expression of E5 albumen often lacks because of HPV genome conformity.Because E5 albumen has high hydrophobicity and more weak antigenicity, and there is no corresponding antibody at present, therefore, obstruction is received to the research of this albumen.Based on the feature of HPVE5 albumen, if a kind of cell strain of the stably express HPV16E5 albumen with label can be built, just a good instrument can be provided for the research of E5 protein biological characteristic and function.Also the desirable cell model becoming the treatment of HPV related neoplasms is hoped.

Summary of the invention

The present invention seeks to utilize recombinant slow virus system to prepare a kind of with label protein and can the cell strain of high expression HPV16E5 albumen, this cell strain provides platform for the biological function of research HPV16E5 albumen, and can be used as the positive control cell strain in the research of HPVE5 albumen.

The establishment method of this cell strain comprises the step building pLVX-Puro-HPV16E5-His recombinant slow virus expression vector, the step of recombinant slow virus packaging, results, transfection Balb/c3T3 cell, through the step that tetracycline (puromycin) screening and RT-PCR, westernblot are identified, the strain of final acquisition stably express HPV16E5 albuminous cell.The cell strain obtained is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCCNo.11794.

The present invention is carrier with recombinant slow virus, is infected Balb/c3T3 mouse embryo cell, and screening obtains can the cell strain of high expression HPV16E5 albumen, this cell strain with the convenient detection being used for HPV16E5 protein expression of His label.

In the step of the structure pLVX-Puro-HPV16E5-His recombinant plasmid described in this invention, first according to HPV16E5 gene design primer, by PCR reaction amplification with the HPV16E5 gene of His label, insert HPV16E5-His gene at EcoR1 and the BamH1 multiple clone site place of pLVX-Puro carrier.Preparing in recombinant slow virus step, utilizing the pLVX-Puro-HPV16E5-His recombinant plasmid that builds and viral packaging plasmid cotransfection 293T cell, through cultivating, going out poison, collect viral supernatants, gather in the crops recombinant slow virus liquid after filtration.Get packaged recombinant slow virus and infect Balb/c3T3 cell, with the Screening of Media containing tetracycline, identify through RT-PCR, westernblot, the Balb/c3T3 cell strain of final acquisition stably express HPV16E5 albumen, build unloaded slow virus simultaneously and transfection Balb/c3T3 cell in contrast.By the frozen preservation in-150 DEG C of refrigerators of cell strain built.

This invention establishes can express the cell strain of HPV16E5 albumen by stability and high efficiency, and this cell strain is that the biological characteristics furtheing investigate HPV16E5 albumen further and the effect causing malignant transformation of cells have established solid foundation.

Accompanying drawing explanation

Fig. 1 pLVX-Puro-HPV16E5-His recombinant plasmid enzyme cuts qualification figure, and wherein 1 is pLVX-Puro-HPV16E5-His recombinant plasmid, 2 be pLVX-Puro empty plasmid in contrast

Fig. 2 pLVX-Puro-HPV16E5-His recombinant plasmid sequencer map

The RT-PCR qualification of Fig. 3 stably expressing cell line, wherein 1 is Balb/c-HPV16E5 cell strain, and 2 is Balb/c-Lenti cell strain in contrast

The Westernblot qualification of Fig. 4 stably expressing cell line

Specific embodiments

(1) structure of recombinant vectors

(1) HPV16E5 gene amplification

According to HPV16 whole genome sequence (GenBankNC_001526); design the HPV16E5 special primer with His label, EcoR1 and BamH1 double enzyme site and protection base, primer sequence is: 5 '-CCGGAATTCGCCACCATGACAAATCTTGATACTGCATCC-3 ' (SEQIDNO.1) and 5 '-CGCGGATCCTTAATGATGATGATGATGATGTGTAATTAAAAAGCGTGCATGT-3 ' (SEQIDNO.2).PCR reaction system is:

(2) DNA agarose gel electrophoresis

The agarose of configuration 1.5%, microwave-oven-heating makes agarose particle dissolve completely; Clean, dry glue plate level is placed on the table; Mixing, encapsulating, inserts comb, avoids producing bubble; After gel sets, carefully take out comb; Glue is put into electrophoresis chamber, adds electrophoretic buffer; By PCR primer loading, through 110V electrophoresis 30min.

(3) PCR primer gel reclaims (QIAquickGelExtractionKit)

Cut the gel of HPV16E5-His gene band, add 3 times of volume QGBuffer and dissolve gel; Hatch 10min in 50 DEG C, rock mixing, until glue dissolves completely; Add 1 times of volume isopropanol precipitation DNA, upset mixing, static 5min; Withdraw mix is on Silica post, and the centrifugal 1min of 13000rpm, discards filtrate; Adding 500 μ lPBBuffer makes DNA be incorporated into Silica post, and the centrifugal 1min of 13000rpm, discards filtrate; Add 750 μ lPEBuffer, the centrifugal 1min of 13000rpm, abandon lower pipe filtrate, the centrifugal 2min of 13000rpm; Renew centrifuge tube, add on EBBuffer to the Silica post of 10 μ l, 50 DEG C of preheatings, the centrifugal 3min of 13000rpm, wash-out obtains DNA.

(4) HPV16E5-His and pLVX-Puro carrier double digestion

HPV16E5-His gel recovery product and pLVX-Puro carrier (being purchased from Clontech company) are carried out BamH1 and EcoR1 double digestion respectively.Enzyme is cut system and is spent the night in 37 DEG C of water-baths, and correct object band is carried out gel recovery by electrophoresis.It is as follows that enzyme cuts system:

(5) HPV16E5 gene is connected with pLVX-Puro carrier

Digestion products HPV16E5-His gene after being reclaimed by gel is connected 4h with pLVX-Puro carrier at 16 DEG C.T4 enzyme linked system is as follows:

Double digestion qualification (see Fig. 1) is carried out to the recombinant plasmid built.

(6) Plastid transformation competence intestinal bacteria Stbl3

Take out E.coliCompetentCellsstbl3 (purchased from Guangzhou FulenGen Co., Ltd.), insert in wet ice and dissolve; 10 μ l are connected product and adds 50 μ l competent cells, rotate mixing gently, ice bath 30min; Heat-shocked intestinal bacteria 45s in 42 DEG C of water-baths, moves into rapidly in wet ice, leaves standstill 2min; Add the SOC nutrient solution 500 μ l of 37 DEG C of preheating antibiotic-frees, cultivate 1h with 150-160rpm rotating speed in 37 DEG C of thermostat container shaking tables.Get the competent cell that 120 μ l have transformed to be applied on the LB agar plate ware containing 100 μ g/ml acillins, flat board is placed in room temperature after its drying, is inverted in incubated overnight 12-16h in the incubator of 37 DEG C, check in each culture dish whether occur bacterium colony.Select the single colony inoculation on agar plate in the LB substratum of 5ml ammonia benzyl resistance, 37 DEG C of shaking table 220rpm overnight incubation, incubation time can not more than 16h.

(7) bacterium liquid object band detects

Bacterium liquid is carried out PCR detection, has the bacterium liquid of object band to send to order-checking (see Fig. 2).The bacterium liquid part checking order correct freezes glycerol stock, preserves for a long time in-80 DEG C.Another part bacterium liquid upgrading grain.

(8) extraction of pLVX-Puro-HPV16E5-His recombinant plasmid

The correct bacterium liquid that checks order carries out plasmid extraction, obtains pLVX-Puro-HPV16E5-His recombinant expression plasmid.Measure OD260 and OD280 with ultraviolet spectrophotometer, calculate nucleic acid purity and nucleic acid content.

(2) acquisition of HPV16E5-Lenti slow virus

(1) packaging of slow virus

Extracting slow virus expression plasmid pLVX-Puro-HPV16E5-His in advance.By the recombinant slow virus plasmid built and virus packaging helper plasmid SL2, SL3, SL4 (being purchased from Clontech company) cotransfection 293T cell (the empty virus vector of transfection pLVX-Puro in contrast).24h before transfection, with the 293T cell of tryptic digestion logarithmic phase, with the substratum adjustment cell density containing 10% serum for 5 × 10 ⁵cell, renewed vaccination 10ml in 10cm Tissue Culture Dish, 37 DEG C, 5%CO ₂in incubator, training 24h, can be used for transfection when cell density reaches 90%-95%.Before transfection, cell culture medium is replaced by serum free medium by 2h.

Prepared pLVX-Puro-HPV16E5-His carrier 4 μ g is added in sterilizing 2ml centrifuge tube, and each 4 μ g of slow virus packaging plasmid SL2, SL3, SL4, mix with the serum-free Opti-MEM substratum of 1.5ml, at room temperature incubation 5 minutes.Get 48 μ lFuGeneHD reagent to mix at another Guan Zhongyu 1.5mlOpti-MEM, incubation at room temperature 5 minutes.Above-mentioned two kinds of solution are mixed, incubation at room temperature 20 minutes, form DNA and FuGeneHD transfection composite.Mixed solution is transferred in the nutrient solution of 293T cell, in 37 DEG C, 5%CO ₂cultivate in cell culture incubator.Remove the substratum containing transfection miscellany after 24h, in every bottle of cell, add the cell culture medium 10ml containing 10% serum, in 37 DEG C, 5%CO ₂continue in incubator to cultivate 24h.The perfect medium changing antibiotic-free after 24h continues to cultivate.

(2) collection of slow virus and concentrated

Collect containing virulent nutrient solution in 50ml sterile centrifugation tube after transfection 48h, then add the fresh complete culture solution of 10ml in the cell that can also produce poison; Again collect containing virulent nutrient solution after transfection 72h, by the nutrient solution of twice collection in 4 DEG C, centrifugal 15min in 3000rpm/min whizzer.Vial supernatant 0.45 μm of membrane filtration removes cell debris, and filtrate, in 4 DEG C, 50000 × g high speed centrifugation 90min, removes supernatant, add 100 μ l complete culture solutions, after slowly fully resuspended with 200 μ l rifle heads, be kept in viral pipe by after the packing of viral concentration liquid, preserve for a long time for-80 DEG C.

(3) slow virus concentration determination

Measure virus titer according to the slow virus dilution metering of standard, finally obtain the slow virus 7.5 × 10 comprising HPV16E5-His gene ⁵tU/ml, unloaded slow virus 5.2 × 10 ⁶tU/ml.

(3) screening of stable transfected cells strain

(1) optimum condition optimization

Slow virus infection method should be optimized according to different clone and the test based on cell.First, the quantity of cell density, slow virus, the parameter such as concentration, infection time of tetracycline are optimized, to determine the optimum condition of each parameter in infection experiment.The infection quantity of slow virus is 10 ²~ 10 ⁶between TU/ml scope, the screening concentration of tetracycline is 5 μ g/mL, namely in 5-7 days, kills all Normocellular concentration.

(2) stable transfected cells strain screening

First day, inoculates 1 × 10 in 6 orifice plates ⁶individual Balb/c3T3 cell, makes next day cell confluency degree reach 80%.Second day, discard substratum, in cell, add the virus liquid (with the cell of the unloaded slow virus of transfection in contrast) diluting suitable multiple with DMEM.The substratum that 2ml is fresh is replaced with after transfection 24h.After transfection 48h, add the cell of the fresh culture screening stable transfection containing 5 μ g/mL tetracyclines.Changed the substratum containing 5 μ g/mL tetracyclines every three days as required, screen about 4 weeks, visible positive cell bunch in 6 orifice plates.Picking positive cell bunch, is seeded in after dilution in 96 orifice plates, guarantees there is a cell in every hole, continues the Screening of Media positive cell strain with purine-containing mycin.

(3) detection of Balb/c-HPV16E5 cell strain

Extract the total serum IgE in positive cell bunch with Trizol, carry out reverse transcription PCR qualification (see Fig. 3).Extract total protein after enlarged culturing is carried out to positive cell, by His monoclonal antibody as primary antibodie, carry out westernblot qualification (see Fig. 4).Finally obtain the Balb/c3T3 cell strain of stable transfection HPV16E5 gene, called after Balb/c-HPV16E5 cell strain, the cell strain obtained is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCCNo.11794, the transfection compared with control cells called after Balb/c-Lenti cell strain of unloaded slow virus.

Claims

1. the cell strain (Balb/c-HPV16E5) of a new stably express HPV16E5 albumen, is characterized in that: this cell strain incorporates HPV16E5 gene, can the expression HPV16E5 albumen of stability and high efficiency.

2. the cell strain (Balb/c-HPV16E5) of a kind of new stably express HPV16E5 albumen as claimed in claim 1,

The preserving number of described cell strain is CGMCCNo.11794.

3. an establishment method for stably express HPV16E5 albuminous cell strain (Balb/c-HPV16E5) as claimed in claim 1, is characterized in that the method comprises the following steps:

1) gene recombination technology is utilized by the HPV16E5 gene clone with His label in Lentiviral;

2) by the recombined lentivirus vector that builds and viral packaging plasmid cotransfection 293T cell, recombinant slow virus is obtained;

3) with recombinant slow virus transfection Balb/c3T3 cell, by the strain of tetracycline screening positive cell;

4) RT-PCR and westernblot qualification is carried out to positive cell strain.

4. construction process obtains the Balb/c-HPV16E5 stable cell line of high expression HPV16E5 albumen according to claim 3.

5. Balb/c-HPV16E5 stable cell line as claimed in claim 4, the preserving number of described cell strain is CGMCCNo.11794.