CN108300729A

CN108300729A - Recombinant plasmid, genetic engineering bacterium containing human cytomegalovirus UL124 genes and its application

Info

Publication number: CN108300729A
Application number: CN201810107622.2A
Authority: CN
Inventors: 朱桦; 曾健雄; 刘奋勇
Original assignee: Jinan University
Current assignee: Jinan University; University of Jinan
Priority date: 2018-02-02
Filing date: 2018-02-02
Publication date: 2018-07-20

Abstract

The invention discloses a kind of recombinant plasmid containing human cytomegalovirus UL124 genes, genetic engineering bacterium and its applications.The recombinant plasmid for containing human cytomegalovirus UL124 genes is obtained by the way that human cytomegalovirus UL124 genes to be inserted into expression plasmid pET32a.Then the prokaryotic expression engineering bacteria of the fusion protein with histidine tag can be expressed by it being rotated into structure in Escherichia coli by heat, which can express corresponding fusion protein.The genetic engineering bacterium for containing human cytomegalovirus UL124 gene recombination plasmids provides raw material for human cytomegalovirus UL124 polyclonal antibodies and monoclonal antibody preparation, simultaneously also be further research UL124 genes concrete function lay the foundation, to for disclose HCMV infection and pathogenic mechanism and treat direction is provided.

Description

Recombinant plasmid, genetic engineering bacterium containing human cytomegalovirus UL124 genes and its Using

Technical field

The present invention relates to field of biology, more particularly to a kind of recombinant plasmid containing human cytomegalovirus UL124 genes, Genetic engineering bacterium and its application.

Background technology

Human cytomegalovirus (human cytomegalovirus, HCMV) is also known as cells inclusions virus, due to infection Cell enlargement, and there is huge intranuclear inclusion, it is therefore named, belong to Betaherpesvirdae, is known maximum DNA blister sores Poison is a kind of opportunistic virion of wide-scale distribution and the important teratogenesis of the mankind, causes neurotrosis pathogen.HCMV is certainly Infect very universal in right crowd, higher in crowd infection rate, the infection rate of developed country is 30~70%.

The infection for the first time of HCMV mostly occurs in infant period.HCMV once invades human body, will be long-term or be present in all the life In vivo, for the normal individual of most immune functions, often it is presented without the latent infection of clinical symptoms；Immune Severe infections can occur in the incomplete baby of systematic growth and immune deficiency person and lethal can die.For pregnant women and youngster The clinical symptoms of child, HCMV infection are more serious, and antenatal merging HCMV acute infections easily lead to fetal abortion, premature labor and abnormal Shape.

It is found in the experiment studied in vitro：Many HCMV genes be for the viral proliferation of itself it is dispensable, because This infer these genes be mainly with virus tissue tropisms, immunologic escape, it is pathogenic, send out, optimizes growth or hide It infects related.

Human cytomegalovirus UL124 genes sequence in HCMV is conservative, and bioinformatic analysis shows that the gene is compiled One transmembrane protein of code.NB, which is tested, detects the transcription product of UL124, after knocking out UL124, HCMV grown in HFF by It seriously undermines, while UL124 is also the relevant transcript of hiding now known related with HCMV latent infection hematopoietic progenitor cells One of.These experimental results and bioinformatic analysis all show that UL124 is extremely important to the infection of HCMV and increment, need into The research of one step.Therefore, the engineering bacteria of human cytomegalovirus UL124 gene recombination plasmids is built, can be to prepare corresponding antibody It lays the first stone, while being also that the further concrete function for studying UL124 genes lays the foundation, to be the infection for disclosing HCMV Being there is provided with pathogenic mechanism and treatment may.

Invention content

The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide one kind and containing human cytomegalovirus The recombinant plasmid of UL124 genes.

Another object of the present invention is to provide the gene works containing above-mentioned human cytomegalovirus UL124 gene recombination plasmids Journey bacterium.

Another object of the present invention is to provide the above-mentioned genetic engineering containing cytomegalovirus UL124 gene recombination plasmids The application of bacterium.

The purpose of the invention is achieved by the following technical solution：A kind of recombination containing human cytomegalovirus UL124 genes Plasmid contains UL124 genetic fragments, wherein the nucleotide sequence of UL124 genetic fragments such as SEQ ID No：Shown in 1.

The construction method of the recombinant plasmid containing human cytomegalovirus UL124 genes by conventional method will UL124 genetic fragments are inserted into pET32a plasmids and obtain.

A kind of genetic engineering bacterium containing human cytomegalovirus UL124 gene recombination plasmids, conversion have above-mentioned huge containing someone The recombinant plasmid of cell virus UL124 genes.

The construction method of the genetic engineering bacterium containing human cytomegalovirus UL124 gene recombination plasmids, for will be upper The recombinant plasmid transformed containing human cytomegalovirus UL124 genes is stated to enter in prokaryotic expression engineering bacteria.

The genetic engineering bacterium is preferably Escherichia coli；More preferably bacillus coli DH 5 alpha or BL21 (DE3).

The genetic engineering bacterium containing human cytomegalovirus UL124 gene recombination plasmids is in UL124 recombinant protein systems Application in standby.

A kind of preparation method of UL124 recombinant proteins, by above-mentioned containing human cytomegalovirus UL124 gene recombination plasmids Genetic engineering bacterium passes through activation culture and fermented and cultured, adds derivant IPTG and carries out induced expression, obtains UL124 and recombinates egg In vain；Preferably：Above-mentioned engineering bacteria is seeded in the LB liquid medium containing ampicillin (Amp) resistance and carries out activation training Support, be then transferred to containing fermented and cultured is carried out in amicillin resistance LB liquid medium, add derivant IPTG into Row induced expression obtains UL124 recombinant proteins.

The condition of the induced expression is preferably：30 DEG C of induction 6h.

The working concentration of the IPTG is 0.5mM/L.

The preparation method of the UL124 recombinant proteins further includes that the UL124 recombinant proteins of acquisition are purified and determined The step of amount：UL124 recombinant proteins are first passed through into nickel column separating purification, BCA methods is then reused and UL124 after purification is recombinated Albumen is quantified.

The genetic engineering bacterium containing human cytomegalovirus UL124 gene recombination plasmids is preparing human cytomegalovirus Application in UL124 protein polyclone antibodies；The engineering bacteria can give expression to the fusion protein with histidine tag.

The present invention has the following advantages and effects with respect to the prior art：The present invention provides one kind containing human cytomegalovirus The engineering bacteria of viral UL124 gene recombination plasmids, by the way that human cytomegalovirus UL124 genes to be inserted into expression plasmid pET32a Recombinant plasmid pET32a-UL124 is obtained, rotates into e. coli bl21 (DE3) to construct to express later by heat and carries The prokaryotic expression engineering bacteria of the fusion protein of histidine tag, the bacterial strain can express corresponding fusion protein, which can To carry out related immunological experiment as antigen, the engineering bacteria of human cytomegalovirus UL124 gene recombination plasmids can be other The structure of the engineering bacteria of related gene recombinant plasmid provides experiment experience, and research is resisted on the material and strategy of human cytomegalovirus More thinking points again.Importantly, providing original for human cytomegalovirus UL124 polyclonal antibodies and monoclonal antibody preparation Material, while being also that the further concrete function for studying UL124 genes lays the foundation, to be the infection and cause for disclosing HCMV Interpretation of the cause, onset and process of an illness system and treatment provide direction.

Description of the drawings

Fig. 1 is the organigram of recombinant plasmid.

Fig. 2 is UL124PCR amplified production electrophoresis schematic diagrames；Wherein, swimming lane M is DNAmarker；Swimming lane 1 and 2 is UL124PCR amplified productions.

Fig. 3 is pET32a electrophoresis result figures；Wherein, figure A and B is the pET32a after pET32a and double digestion；Swimming lane M is DL5000DNAMarker；It is pET32a (5900bp) to scheme swimming lane 1 and 2 in A；It is pET32a double digestions to scheme swimming lane 1 and 2 in B.

Fig. 4 is bacterium colony PCR and extraction plasmid double digestion electrophoresis schematic diagram.Wherein, figure A is the bacterium colony converted after attachment PCR；Figure B is the double digestion qualification result for extracting plasmid；Swimming lane M is DL2000DNA Marker；Swimming lane 1 and 2 is in figure A UL124 bacterium colonies PCR (459bp)；Swimming lane 1 and 2 is the double digestion qualification result for extracting plasmid in figure B.

Fig. 5 is the optimum results figure of induced expression condition；Wherein, figure A is the optimum results of induced expression temperature, swimming lane M For albumen Marker, swimming lane 1 is to be not added with derivant, and swimming lane 2,3,4,5 is respectively 16 DEG C, 20 DEG C, 30 DEG C, 37 DEG C of inducing temperature (IPTG concentration 0.5mmol/L, induction time 6h)；Scheme the optimum results that B is induced expression IPTG concentration, swimming lane M is albumen Marker, swimming lane 1 are to be not added with derivant, the respectively IPTG concentration 0.1mmol/L, 0.3mmol/L of swimming lane 2,3,4,5,6,7, 0.5mmol/L, 0.8mmol/L, 1.0mmol/L, 1.5mmol/L (30 DEG C of inducing temperature, induction time 6h)；It is induction to scheme C The optimum results of expression time, swimming lane M are albumen Marker, and swimming lane 7 is to be not added with derivant, and swimming lane 1,2,3,4,5,6 is respectively Induction time 12h, 10h, 8h, 6h, 4h, 2h (IPTG concentration 0.5mmol/L, 30 DEG C of inducing temperature).

Fig. 6 is purifying schematic diagram and the fusion of the expression bacterium expressed fusion protein containing recombinant plasmid pET32a-UL124 The Western blot result figures of albumen；Wherein, figure A is the expression bacterium expression fusion egg containing recombinant plasmid pET32a-UL124 White purifying schematic diagram, each swimming lane (swimming lane 1~8) from left to right indicate albumen Marker, are pierced by, 25mmol/L imidazoles respectively Eluent, 100mmol/L imidazole elutions, 150mmol/L imidazole elutions, 200mmol/L imidazole elutions, 500mmol/L Imidazole elution, 500mmol/L imidazole elutions；Scheme the Western blot result figures that B is fusion protein；Swimming lane M is albumen Marker；Swimming lane 1 is the total bacterial protein for being not added with derivant in figure B, and 2 be UL124 fusion proteins.

Specific implementation mode

With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.

Embodiment 1

One, plasmid construction

(1) UL124 gene primers design

According to the sequence of UL124 genes in TB40/E (KF297339.1) the Strain genome announced on GenBank, entirely Long 459bp (gene order such as SEQ ID No：Shown in 1；Its protein translation product such as SEQ ID No：Shown in 2), be with the sequence Template, it is soft using Primer Select with PET32a plasmids (excellent precious biological Co., Ltd) for cloned plasmids and expression plasmid Part chooses suitable restriction enzyme site and carries out design of primers.Designed primer such as following table：

1 UL124 design of primers tables of table

Note：F (sense primer)；R (downstream primer).

(2) UL124 gene PCRs

According to the gene order (GenBank for the HCMV TB40/E-BAC being published on GenBank：EF999921.1, Quote article (Cloning and sequencing of a highly productice, endotheliotropic virus Strain derived from human cytomegalovirus TB40/E)) it is template, it is carried out in following reaction system PCR：

2 PCR reaction system tables of table

PCR amplification condition see the table below：

Table is arranged in 3 PCR reaction conditions of table

Take 5 μ l of PCR product into row agarose gel electrophoresis, according to glue interpretation of result is run, if band specificity is very high, It can use PCR product purification kit recovery product that must terminate PCR product whole loading electrophoresis, electrophoresis if having miscellaneous band appearance Afterwards, adhesive tape of corresponding size is cut according to Maker, is recycled using omega plastic recovery kits.

Two, UL124 genes agarose gel electrophoresis

1, according to the size of the target DNA fragment expanded, the Ago-Gel of suitable concentration is selected；UL124 genes are complete Long 459bp base-pairs select 1% Ago-Gel；

2, agarose powder 0.2g is weighed, the 1 × TAE buffer solution 20mL for preparing and having diluted in advance are added, are put in microwave In stove, moderate heat heats 2min；

3, when the gel room temperature melted is cooled to 60 DEG C or so, the GelStain of 0.2 μ l is added thereto, fully shakes up It slowly pours into and is plugged in the glue groove of comb in advance afterwards, bubble is avoided to generate, after being stored at room temperature about 30min, gel can coagulate completely Gu glue is completed；

4, start before running glue, gently pull up comb from vertical direction, gel is put into electrophoresis tank, ensure the leaching of electrophoresis liquid energy Do not have monoblock glue, after placing steadily, glue can be run by the sequence point sample finished in advance；

5, after point sample, according to target DNA size and gel strength, electrophoretic parameters are set as：Electric current 200mA, time 45min (runs glue to adjust according to the position of bromophenol blue and run the glue time in the process)；

6, after completing electrophoresis, gel is taken out from electrophoresis tank, and electrophoresis liquid is filtered dry, be put in preprepared preservative film On, it is placed into gel imager, adjusts suitable angle, take pictures, analyze and preserve result (Fig. 2).

Three, UL124 genes gel recycles

1, it after vertical agarose gel electrophoresis, moves in dark place, under ultra violet lamp, according to Maker and purpose Mrna length cuts the Ago-Gel containing target DNA with clean blade；

When 2, cutting target DNA, as possible precisely, the extra gel except target DNA band is cut less, it is suitable by what is cut When the EP that the gel piece of size is put into a sterile 1.5ml of preprepared manages (having weighed), after weighing, calculation takes gel Net weight, the addition XP2 solution of equal proportion volume；

3, the EP pipes of the 1.5ml for the gel piece for being mixed with XP2 solution are placed in water-bath, 57 DEG C of heating, every 2~3min Reverse mixing, until gel piece melts completely；

Its mediation mixing and of short duration centrifugation, can finally be added into advance by 4, the gel cooling after complete melt Assembled silicagel column, room temperature, 10000 × g centrifuge 1min, topple over the waste liquid in collecting pipe；

5, it adds in 300 μ l XP2 solution to silicagel column, after room temperature 10000 × g centrifugations 1min, by the liquid in collecting pipe Body is outwelled；

6, it washs solution with 700 μ l SPW and washs silicagel column, 10000 × g of room temperature centrifuges 1min, by the liquid in collecting pipe It outwells；

7, it is primary that 6 steps are repeated；

8, sky gets rid of silicagel column, and 12000 × g of room temperature centrifuges 2min, removes extra SPW washing solution；

9, it takes out silicagel column and puts it into the 1.5mL EP pipes of sterilizing, after being placed at room temperature for 2min, into silicagel column 30 μ l are added and have been heated to 65 DEG C of sterile ddH20 in advance, after being stored at room temperature 1min, 12000 × g centrifuges 1min, and taking-up has been discarded Silicagel column, the liquid collected in EP pipes is target DNA solution；

10, the target DNA concentration and purity of detection recycling, sample are put in -20 DEG C of preservations.

Four, pET32a plasmid extractions

1, the plasmid evening before yesterday is taken out, the DH5 α strains containing pET32a plasmids is taken out from -20 DEG C of refrigerators, draws 4 μ l, be inoculated in and contain In the 4mL LB liquid mediums of ampicillin (final concentration of 100 μ g/ml), 220rpm/min cultures 12 in 37 DEG C of incubators ~16h；

2, second day, take out the bacterium solution that has been incubated overnight, and thalline is added in 1.5mL EP pipes, 12000 × g room temperatures from Heart 1min abandons supernatant, repeats step, 4mL bacterium solutions are all collected into 1.5mL EP pipes；

3, it exhausts after extra supernatant, adds in 250 μ l solution I to EP pipes, it is heavy that thalline is gently blown and beaten with pipettor It forms sediment, after thalline resuspension, is put on the rotary vibrator of whirlpool, makes its complete mixing；

4,250 μ l solution II are added, for several times, the reverse time will be got hold of the mixing that lightly turns upside down, control System is between 2min~5min, and after having overturned, liquid generally can transparent clarification；

5, be eventually adding 350 μ l solution III, the mixing that lightly turns upside down for several times, until there is white precipitate to be formed, Reverse mixing number cannot be excessive, is and then put in compact centrifuge, room temperature, and 13000 × g centrifuges 10min；

6, after centrifuging, liquid-transfering gun is transferred to supernatant in marked good silicagel column, silicagel column puts 2ml's into In centrifuge tube, white depositions are not sucked in silicagel column, are put in compact centrifuge, 10000 × g of room temperature, centrifuges 1min；

7, the waste liquid in collecting pipe is outwelled, 500 μ l HB solution is drawn, is added in silicagel column, room temperature 10000 × g centrifugations 1min；

8, the waste liquid in collecting pipe is outwelled, adds in 700 μ l DNAWash Buffer to silicagel column, (first checks DNAWash Whether Buffer has been added absolute ethyl alcohol) room temperature 10000 × g centrifugations 1min；

9, it is primary that 8 steps are repeated；

10, sky gets rid of silicagel column, and 13000 × g of room temperature centrifuges 2min, removes extra DNAWash Buffer；

11, it takes out silicagel column to put it into the 1.5mL EP pipes of sterilizing, 50 μ l is added into silicagel column and have added in advance Heat is to 65 DEG C of sterile ddH20, and after being stored at room temperature 1~2min, 12000 × g centrifuges 1min, takes out obsolete silicagel column, EP pipes Middle liquid is target DNA solution；

12,1 μ l sample detections are taken to extract the concentration and purity of plasmid, remaining sample is for subsequent experimental or is put in -20 DEG C preserve.

Five, double digestion

1, UL124PCR products double digestion system such as following table：

4 PCR product double digestion system table of table

Ingredient	Volume (total 20 μ l)
		UL124PCR products	10μl
10×FastDigest buffer	2μl
		FastDigest BamHI	1μl
FastDigest HindIII	1μl
		ddH2O	6μl

Note：Endonuclease reaction condition：Digestion 50min in 37 DEG C of thermostat water baths；Control digestion system DNA total amounts are less than or wait In 1 μ g.

2, it needs to be purified after completing endonuclease reaction.

3, pET32a plasmids double digestion system such as following table：

5 pET32a plasmid double digestion system tables of table

Note：Endonuclease reaction condition：Digestion 50min in 37 DEG C of thermostat water baths；Control digestion system plasmid total amount be less than or Equal to 1 μ g.

4, it needs to be purified after completing endonuclease reaction.

Six, Product Purification Kit recycles double digestion product

1,4~5 times of combination liquid CP is added according to double digestion system, mixes well；

2, previous step acquired solution is added in adsorption column EC, is placed at room temperature for 1min, 10000 × g, centrifuged 1min, outwell Waste liquid in collecting pipe；

3,700 μ l rinsing liquids WB (first check whether and absolute ethyl alcohol has been added), 10000 × g are added, centrifuges 1min, discards Waste liquid；

4, it is primary that 3 steps are repeated；

5, adsorption column EC is put back in collecting pipe, 10000 × g, centrifuges 1min, removes rinsing liquid as far as possible, in order to avoid residual Ethyl alcohol inhibits downstream reaction；

6, adsorption column is taken out, is put into a clean centrifuge tube, adds 30 μ l elution buffers at the intermediate position of adsorbed film Liquid EB (heating effect is more preferable in 65~70 DEG C of water-baths in advance for elution buffer) is placed at room temperature for 2min, 13000 × g centrifugations 1min.If necessary to more amount DNA, obtained solution can be rejoined in adsorption column, centrifuge 1min.Take out obsolete silicon Rubber column gel column, the liquid collected in EP pipes is target DNA solution；

7, the target DNA concentration and purity of detection recycling, to be preferably used for subsequent experimental.Sample is put in -20 DEG C of preservations. Experimental result is as shown in Figure 3, wherein figure A and B is the pET32a after pET32a and double digestion.

Seven, UL124 double digestions recovery product connect reaction with pET32a plasmid double digestion recovery products

Coupled reaction system such as following table：

6 coupled reaction system table of table

Ingredient	Volume (total 10 μ l)
		PET32a plasmids	5μl
UL124	2μl
		10×T4Ligation Buffer	2μl
T4DNA Ligase	1μl
		ddH2O	0μl

Connection reaction condition is to be placed in connection 16h or more in 16 DEG C of connection instrument, obtains recombinant plasmid.

Eight, the preparation of bacillus coli DH 5 alpha competent cell

1, it one lives：The unloaded strain DH5 α preserved with the activation of LB culture mediums, by volume 1：100 are inoculated with, 37 DEG C of perseverances Warm shaking table 220rpm/min activates 12~16h；

2, it two lives：Strain DH5 α after being lived with LB culture mediums activation one, by volume 1：100 are inoculated with, 37 DEG C of constant temperature Shaking table 220rpm/min activation makes OD600 values be 0.4~0.6；

3, take the bacterium solution 1ml after two work in being placed in ice bath 10min on ice in EP pipes, according to actual needs prepared by quantity；

4,4 DEG C, 4000 × g, most supernatant is abandoned as far as possible after centrifuging 5min；

2, the CaCl of the 1ml 0.1M that ice bath (30min) has been crossed is added₂Thalline, then ice bath 30min is resuspended；

6, it 4 DEG C, 4000 × g, centrifuges and abandons most supernatant as far as possible after 5min (dried filter paper will be remaining after can using sterilizing Liquid exhausts)；

7, the CaCl of 50 μ l 0.1M is added₂Thalline is resuspended, can be used after ice bath 0.5h, this is the DH5 α prepared Competent cell；

8, the competence bacteria DH5 α prepared -20 DEG C are placed on to freeze and (used in one month).

Nine, it converts

1, volume aspirated is that the connection liquid of 5 μ l is added in the DH5 α competent cells of the 50 μ l prepared in advance, gently Mixing is positioned on ice, ice bath 30min；

2, it after the completion of waiting for ice bath, will be put in 42 DEG C of preheated in advance thermostat water baths added with the competence of connection liquid, Heat shock 90s；

3, after completing heat shock, immediately competence is positioned on ice, ice bath 2min；

4, after ice bath 2min, the LB liquid medium of 900 μ l room temperatures is added into every EP pipe,

37 DEG C of constant-temperature shaking incubator concussions are put in, rotating speed 180rpm/min cultivates 45min；

5, cultured bacterium is put in centrifuge, room temperature, 3000 × g, time 60s after centrifugation, carefully inhales 700 μ l supernatants are removed, about 100 μ l culture mediums are stayed, thalline is resuspended；

6, the thalline for drawing 100 μ l is coated on and pre-prepared has added ampicillin (final concentration of 100 μ g/ml) anti- Property LB tablets on, be placed in 37 DEG C of constant incubators, after just setting 15min, wait for that bacterium solution is absorbed completely, be inverted culture, culture Time is 12~16h.

Ten, bacterium colony PCR is identified

1, it from 2 monoclonal colonies of picking on the LB tablets of the amicillin resistance with monoclonal, is inoculated into respectively In 4ml LB liquid mediums containing ampicillin, 37 DEG C, rotating speed 220rpm/min, isothermal vibration incubator culture, training It is 12~16h to support the time；

2, from cultured bacterium solution, masterplates of the 5 μ l as bacterium colony PCR is drawn, PCR amplification identification, specific body are carried out System see the table below：

7 bacterium colony PCR reaction system tables of table

Reagent name	Volume (total 25 μ l)
		Sterile ddH2O	13.25μl
10×PCR buffer(with Mg2+)	2.5μl
		dNTP	2μl
Bacterium solution template	5μl
		Sense primer	1μl
Downstream primer	1μl
		TaKaRa Taq	0.25μl

PCR amplification condition see the table below：

Table is arranged in 8 bacterium colony PCR reaction conditions of table

3, after PCR amplification, reaction product runs 1% agarose gel electrophoresis, and gel imager is taken pictures, and analyze, Preserve experimental result.Experimental result is as shown in Figure 4 A.

11, double digestion is identified

1, according to the qualification result of bacterium colony PCR, the bacterium solution of target DNA fragment size will be amplified, carries out conservation, conservation Method be total volume 1ml, bacterium solution 750 μ l, 100% 250 μ l of glycerine, mixing place -20 DEG C of preservations, and remaining bacterium solution is equal It is used for extract plasmid；

2, the recombinant plasmid for obtaining extracting carries out double digestion identification, and reaction system see the table below：

9 recombinant plasmid double digestion reaction system table of table

Ingredient	Volume (total 20 μ l)
		PET32a plasmids (contain Insert Fragment)	10μl
10×FastDigest buffer	2μl
		FastDigest BamHI	1μl
FastDigest HindIII	1μl
		ddH2O	6μl

Note：Endonuclease reaction condition：Digestion 50min in 37 DEG C of thermostat water baths.

3,1% Ago-Gel is run after double digestion, is taken pictures, and result is analyzed and preserve.Experimental result is as shown in Figure 4 B.

12, Plasmid samples are sequenced

Company is sent to be sequenced the Plasmid samples that can go out target gene size segment with double digestion, it, will after sequencing result comes out Sequencing result is compared with the gene order above GennBank using BLAST, analyses and comparison result.If sequencing result with Download sequence mismatches, and repeats the above experiment until sequencing result matches.

13, (method refers to the 8th " system of bacillus coli DH 5 alpha competent cell for the preparation of competence bacteria BL21 (DE3) It is standby ").

14, pET32a (UL124) conversion expression bacterium BL21 (DE3) (method refers to the 9th " conversion ").

15, the prokaryotic expression (DH5 α) of UL124 recombinant proteins

(1) UL124 recombinant protein expression quantity is tested in different temperatures culture

1, experiment is provided with 16 DEG C, and 20 DEG C, 30 DEG C, 37 DEG C of 4 temperature are expressed, and volume of culture condition setting is：4ml Liquid LB Tube propagations, final concentration 0.5mmol/L, the induction time 6h of IPTG.

2, above expression thalline 1ml, 10000 × g are collected, most supernatant is abandoned as far as possible after centrifuging 1min, is separately added into 60 μ l 1 × SDS-PAGE Loading Buffer, with liquid-transfering gun blow and beat mixing, after 100 DEG C of the above sample boils 10min, -20 DEG C Preserve or run the analysis of SDS-PAGE protein adhesives；

3, after the analysis of SDS-PAGE protein adhesives, the best 30 DEG C of conducts of temperature of expression effect in above 4 temperature are determined Express temperature (Fig. 5 A).

(2) various concentration IPTG expresses UL124 recombinant proteins and tests

The experiment is carried out using the inducing temperature of the above single factor experiment, setting IPTG induced concentrations are：0mM/L, 0.1mM/L, 0.3mM/L, 0.5mM/L, 0.8mM/L, 1.0mM/L, 1.5mM/L, 4ml liquid LB Tube propagations are expressed, induction temperature 30 DEG C of degree, induction time 6h.

Most supernatant is abandoned as far as possible after collecting above expression thalline 1ml, 10000 × g centrifugation 1min, is separately added into the 1 of 60 μ l × SDS-PAGE Loading Buffer blow and beat mixing with liquid-transfering gun, after 100 DEG C of the above sample boils 10min, -20 DEG C of preservations Or run the analysis of SDS-PAGE protein adhesives.According to experimental result, determine that the above various concentration derivant IPTG expression effects are best Concentration 0.5mM/L be derivant best induced concentration (Fig. 5 B).

(3) experiment of the different incubation times to UL124 recombinant protein expression quantity

1, the experiment being carried out using the inducing temperature of the above single factor experiment and inducer concentrations, 2h, 4h is arranged in experiment, Totally 6 time points collect thalline by 6h, 8h, 10h, 12h；4ml liquid LB Tube propagations are expressed, IPTG concentration 0.5mmol/L, induction 30 DEG C of temperature.

2, above expression thalline 1ml, 10000 × g are collected respectively, are abandoned most supernatant as far as possible after centrifuging 10min, are separately added into 1 × SDS-PAGE Loading Buffer of 60 μ l blow and beat mixing with liquid-transfering gun, after 100 DEG C of the above sample boils 10min ,- 20 DEG C preserve or run the analysis of SDS-PAGE protein adhesives.

3, after the analysis of SDS-PAGE protein adhesives, the best induction expression protein acquisition time point in above 6 times is determined For 6h (Fig. 5 C).

16, UL124 recombinant proteins are collected

1, it determines that expression temperature, inducer concentrations collect the thalline time according to the above experiment, expands the expression body of expression bacterium Product collects thalline, and 4 DEG C, 10000 × g abandons most supernatant as far as possible after centrifuging 10min；

2, thalline is resuspended in 1 × PBS buffer solution that (1/20 cell growth volume) is added, and 4 DEG C, 10000 × g, centrifuges 10min Most supernatant is abandoned as far as possible；

3, precipitation is added 1 × PBS buffer solution and thalline is resuspended, and carrying out ice-bath ultrasonic wave using sonicator breaks bacterium, breaks Bacterium parameter is set as：150W, work 4s, stops 6s, is crushed about 30min, until bacterium solution becomes clarification；

4,10000 × g, centrifuges 30min by 4 DEG C, and supernatant, precipitation is gone to be resuspended and washed with 1 × PBS buffer solution, 10000 × g, and 4 DEG C, 30min is centrifuged, supernatant is removed.

5, urea liquid of the precipitation containing 8mol/l dissolves 1h, centrifuges to obtain supernatant again, with 0.22 μm of membrane filtration, into Row protein purification.

17, the isolation and purification of recombinant protein

1, Ni columns are cleaned with the deionized water of 10 times of column volumes (0.22 μm of membrane filtration)；

2, pillar is balanced with the 5mmol/l imidazole buffers (containing 8mol/l urea) of 5 times of column volumes；

3, loading, coutroi velocity are pierced by liquid in 1ml/min or so, collection, are analyzed for SDS/PAGE protein adhesives；

4, contain 20mmol/l, 100mmol/l, 150mmol/l, 200mmol/l, 500mmol/l with 5 times of column volumes respectively The buffer solution of imidazoles elutes, and flow control collects eluent in 2ml/min, and the eluent that keeps sample respectively is for SDS/PAGE points Analyse the distribution situation of protein.

Experimental result：Containing recombinant vector pET32a-UL124 expression bacterium expressed fusion protein purifying schematic diagram and The results are shown in Figure 6 by the Western blot of fusion protein.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications, Equivalent substitute mode is should be, is included within the scope of the present invention.

Sequence table

<110>Ji'nan University

<120>Recombinant plasmid, genetic engineering bacterium containing human cytomegalovirus UL124 genes and its application

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 459

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>UL124 genes

<400> 1

atggaaagga acagtctgtt agtctgtcag ctattatgtc tggtggcgcg cgcggcagca 60

acgagtactg ctcagactac actgccctcc accgttaaca gcaccgcaac gggagttacc 120

tctgactctt atcagaacac aacaactcag ctgcctgcat cttcttctgc cgctgcctta 180

agtcttccaa atgcgtcagc ggtgcaagcc cgctccccga gctcattttc agacacatac 240

cctaccgcca cggccttgtg cggcacactg gtggtggtgg gcatcgtgct gtgcctaagt 300

ctggcctcca ctgttaggag caaggagctg ccaagcgacc atgagccgct ggaggcatgg 360

gagcagggct cggatgtaga agctccgccg ctaccggaga agagcccatg tccggaacac 420

gtacccgaga ttcgcgtgga gatcccacgc tatgtttaa 459

<210> 2

<211> 152

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>UL124 protein translation products

<400> 2

Met Glu Arg Asn Ser Leu Leu Val Cys Gln Leu Leu Cys Leu Val Ala

1 5 10 15

Arg Ala Ala Ala Thr Ser Thr Ala Gln Thr Thr Leu Pro Ser Thr Val

20 25 30

Asn Ser Thr Ala Thr Gly Val Thr Ser Asp Ser Tyr Gln Asn Thr Thr

35 40 45

Thr Gln Leu Pro Ala Ser Ser Ser Ala Ala Ala Leu Ser Leu Pro Asn

50 55 60

Ala Ser Ala Val Gln Ala Arg Ser Pro Ser Ser Phe Ser Asp Thr Tyr

65 70 75 80

Pro Thr Ala Thr Ala Leu Cys Gly Thr Leu Val Val Val Gly Ile Val

85 90 95

Leu Cys Leu Ser Leu Ala Ser Thr Val Arg Ser Lys Glu Leu Pro Ser

100 105 110

Asp His Glu Pro Leu Glu Ala Trp Glu Gln Gly Ser Asp Val Glu Ala

115 120 125

Pro Pro Leu Pro Glu Lys Ser Pro Cys Pro Glu His Val Pro Glu Ile

130 135 140

Arg Val Glu Ile Pro Arg Tyr Val

145 150

<210> 3

<211> 33

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>UL124 primers-F

<400> 3

cgcgcgaagc ttaacatagc gtgggatctc cac 33

<210> 4

<211> 39

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>UL124 primers-R

<400> 4

cggcgcggat ccatggaaag gaacagtctg ttagtctgt 39

Claims

1. a kind of recombinant plasmid containing human cytomegalovirus UL124 genes, it is characterised in that：Containing UL124 genetic fragments, In, the nucleotide sequence such as SEQ ID No of UL124 genetic fragments：Shown in 1.

2. a kind of genetic engineering bacterium containing human cytomegalovirus UL124 gene recombination plasmids, it is characterised in that：Conversion is had the right It is required that the recombinant plasmid containing human cytomegalovirus UL124 genes described in 1.

3. the genetic engineering bacterium according to claim 2 containing human cytomegalovirus UL124 gene recombination plasmids, feature It is：The genetic engineering bacterium is Escherichia coli.

4. the genetic engineering bacterium according to claim 2 containing human cytomegalovirus UL124 gene recombination plasmids, feature It is：The genetic engineering bacterium is bacillus coli DH 5 alpha or BL21 (DE3).

5. the genetic engineering bacterium that claim 2~4 any one of them contains human cytomegalovirus UL124 gene recombination plasmids exists Application in the preparation of UL124 recombinant proteins.

6. a kind of preparation method of UL124 recombinant proteins, it is characterised in that：Claim 2~4 any one of them is contained into someone The genetic engineering bacterium of cytomegalovirus UL124 gene recombination plasmids passes through activation culture and fermented and cultured, adds derivant IPTG carries out induced expression, obtains UL124 recombinant proteins.

7. the genetic engineering bacterium that claim 2~4 any one of them contains human cytomegalovirus UL124 gene recombination plasmids exists Prepare the application in human cytomegalovirus UL124 protein polyclone antibodies.