CN103923938A

CN103923938A - Cloning and expression of beta-subunit gene of phycocyanin

Info

Publication number: CN103923938A
Application number: CN201410186005.8A
Authority: CN
Inventors: 欧瑜; 张静
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2014-04-30
Filing date: 2014-04-30
Publication date: 2014-07-16

Abstract

The invention belongs to the technical field of genetic engineering and particularly relates to cloning and expression of a beta-subunit gene of phycocyanin. The beta-subunit gene of phycocyanin is cloned to a pET-32a plasmid vector by a genetic engineering means, and the recombinant plasmid is converted into competent escherichia coli BL21. The expression product constructed by engineering bacteria is purified by a nickel affinity column, and the obtained fusion protein has a certain inhibiting effect on proliferation of Hela cells.

Description

The clonal expression of phycocyanin beta subunit gene

Technical field

The invention belongs to gene engineering technology field, be specifically related to the clonal expression of phycocyanin beta subunit gene.

Technical background

Phycocyanins, C-(phycocyanin, PC) is that one is present in blue-green algae and the intracellular photosynthetic pigments of red algae, can efficient capture luminous energy.Its basic building unit is α and β subunit, and molecular weight is between 17kD to 22kD.α subunit is made up of 162 amino-acid residues, and β subunit is made up of 172 amino-acid residues.Research finds, that the physiologically active of Phycocyanins, C-comprises is antitumor, anti-oxidant, anti-inflammatory, raising body capability of resistance to radiation, neuroprotective tissue are avoided damage, strengthen immunologic function, inhibition haemolysis etc.By the β subunit of Phycocyanins, C-at expression in escherichia coli, find that there is antitumour activity in recombinant chou phycocyanin beta unit, but in normal cell, have very little reaction, anticancer propagation and cell death inducing may make recombinant chou phycocyanin beta unit become the means of a kind of effective cancer prevention and treatment.Research finds that β subunit has stronger restraining effect to the growth of some tumour cell compared with the Phycocyanins, C-and α subunit thereof of same molar ratio, and prompting β subunit has the good potentiality that becomes antitumor drug.

The present invention is the gene of clonal expression phycocyanin beta subunit first, and the fusion rotein of purifying expression, and the restraining effect of this albumen of vitro detection to Hela Growth of Cells, for its research and development as antitumor drug provide experimental basis.

Summary of the invention

Phycocyanin beta subunit fusion rotein of the present invention is by being transformed into competence intestinal bacteria after construction of recombinant plasmid, by abduction delivering, and purifying gained albumen and obtaining.Its preparation method is as follows:

The recombinant plasmid that contains the whole β subunits of Phycocyanins, C-and part α subunit gene of preserving from this laboratory, PCR method amplifies beta subunit gene fragment, with restriction endonuclease BamH I, HindIII37 DEG C double digestion beta subunit gene fragment and pET-32a plasmid 1.5h, after agarose gel electrophoresis, cut glue and reclaim object product 16 DEG C of connection 16h again.Connecting product transforms in competent escherichia coli cell BL21.Engineering bacteria is the logarithmic phase to bacterial growth by LB culture medium culturing, and adding final concentration is the IPTG of 0.2mmol/L, 20 DEG C of abduction delivering 20h.Induction finishes the centrifugal collection thalline of rear 10000rpm, 15min, add the ratio of 50ml damping fluid according to every collection 1L thalline, with the resuspended thalline of damping fluid, 60% power ultrasonic fragmentation, 12000rpm, 30min collect supernatant, obtain the fusion rotein of phycocyanin beta subunit with nickel affinity chromatography column purification.

Fusion rotein containing phycocyanin beta subunit of the present invention is acted on to Hela cell, and mtt assay detects its restraining effect to Hela cell proliferation, finds that this fusion rotein has obvious inhibited proliferation to Hela cell.

Brief description of the drawings

Fig. 1 is the phycocyanin beta subunit gene order that pcr amplification goes out.M:DNAMarker; 1: phycocyanin beta subunit gene.

Fig. 2 is the phycocyanin beta subunit gene order that bacterium liquid pcr amplification goes out.M:DNA Marker; 1: phycocyanin beta subunit gene.

Fig. 3 is the recombinant plasmid double digestion checking of extracting.M:DNAMarker; 1: the phycocyanin beta subunit gene that double digestion obtains.

Fig. 4 is fusion rotein nickel affinity chromatography column purification result.M: albumen marker; 1: thalline before induction; 2: thalline after induction; 3: the centrifugal rear supernatant of thalline ultrasonication; 4: thalline ultrasonication centrifuged deposit; 5: fusion rotein nickel affinity chromatography column purification effect.

Embodiment

Below in conjunction with some examples and with reference to chart data, the present invention is described further.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.

Embodiment 1

Contain the structure of the recombinant plasmid of phycocyanin beta subunit gene

Select the gene order clone that phycocyanin beta subunit is corresponding, sequence length is 519bp.In the upstream and downstream primer of goal gene, add respectively BamH I, HindIII restriction enzyme site.The recombinant plasmid pMD18-T-PC containing the whole β subunits of Phycocyanins, C-and part α subunit gene preserving with this laboratory is as template, the corresponding gene of the performing PCR of going forward side by side amplification β subunit.After PCR reaction finishes, product carries out 2% agarose gel electrophoresis, as Fig. 1, cuts the gel that contains target DNA, reclaims test kit specification sheets reclaim target DNA fragment according to DNA gel.Goal gene fragment and pET-32a plasmid vector be double digestion simultaneously, gets double digestion product and carries out 2% agarose gel electrophoresis, cuts the gel that contains target DNA, reclaims test kit specification sheets reclaim object fragment according to gel.Goal gene fragment and prokaryotic expression carrier pET-32a after double digestion are connected under 16 DEG C, 16h condition.

Embodiment 2

Recombinant plasmid transformed is in competence e. coli bl21

Get connection product 10 μ l and join the e. coli bl21 competent cell that 50 μ l have prepared, mix gently rear ice bath 30min; In 42 DEG C of water-baths after heat shock 90s, ice bath 3min again rapidly; Add 500 μ l LB liquid nutrient mediums and fully mix, 37 DEG C, 220rpm jolting cultivation 1h; The centrifugal supernatant of abandoning is left and taken approximately 100 μ l transformed bacteria liquid, coats containing penbritin (Amp ⁺, 100 μ g/mL) LB solid medium flat board on, 37 DEG C cultivate 12-16h.Picking transforms successful mono-clonal and carries out bacterium liquid PCR checking (Fig. 2), extracts plasmid double digestion checking (Fig. 3), and order-checking.

Embodiment 3

The abduction delivering of engineering bacteria

The bacterial classification of preservation is inoculated in the small test tube containing LB substratum, and it is saturated that 37 DEG C, 220rpm shake to bacterium liquid, as kind of a daughter bacteria.Saturated bacterium liquid is transferred in the Erlenmeyer flask containing LB substratum in 1:100 ratio, and 37 degree 220rpm shake OD600 between 0.6-0.8.Adding final concentration is the IPTG of 0.2mmol/L, 20 DEG C of induction 20h.The centrifugal collection thalline of 10000rpm, 15min.

Embodiment 4

The purifying of fusion rotein

The thalline of collecting is resuspended with damping fluid, ultrasonication thalline, and 12000rpm, 30min collect supernatant.Nickel ion affinity chromatograph column purification albumen: with binding buffer liquid balance pillar, will flow to end time, will add in post with the ultrasonic supernatant liquor after 0.45 μ m filtering with microporous membrane; Treat that supernatant liquor flows to end, rinse filler with binding buffer liquid and wash away unconjugated albumen; Finally, with containing the elution buffer wash-out target protein of 200mM imidazoles, collect elutriant concentration, to add final concentration be 10% glycerine and filter rear for subsequent use by 0.22 μ m sterile filters.

Embodiment 5

The proliferation inhibition test of fusion rotein to Hela cell

The fusion rotein containing phycocyanin beta subunit after purifying is acted on to cervical cancer cell Hela, final concentration is respectively the fusion rotein of 0,0.625,1.25,2.5,5,10 μ mol/L and the pET-32a empty carrier albumen of 10 μ mol/L, after 72h, mtt assay detects Hela cell survival rate.Concrete operation step: get well-grown logarithmic phase Hela cell, making concentration with DMEM substratum is 2 × 10 ⁴the single cell suspension of individual/mL, every hole adds 100 μ l to be cultured to cell attachment in 96 orifice plates.Blank is established in experiment: for zeroing; Negative control group: the pET-32a empty carrier albumen that only adds 10 μ mol/L; Experimental group: 0.625, the fusion rotein group containing phycocyanin beta subunit of 1.25,2.5,5,10 μ mol/L, every hole adds substratum to cumulative volume 100 μ l.In 37 DEG C of CO2gas incubator, cultivate abandoning supernatant after 72h, add the MTT of 5mg/mL, every hole 10 μ l, 37 DEG C are continued to cultivate 4h.Abandon supernatant liquor, add DMSO150 μ l/L hole, after vibration mixes, in microplate reader, measure the OD value under 570nm, be calculated as follows fusion rotein to Hela inhibitory rate of cell growth:

Inhibiting rate %=(1-OD _{experimental group}/ OD _{negative control group}) × 100%

Table 1

Result confirms by experiment, and phycocyanin beta subunit fusion rotein is in the time of 10 μ mol/L concentration, and it is 32.52% to the proliferation inhibition rate of Hela cell that mtt assay records 72h.

Claims

1. the clone of phycocyanin beta subunit gene on plasmid vector, is characterized in that: plasmid vector is pET-32a, and restriction enzyme site is BamH I, HindIII.

2. recombinant plasmid according to claim 1, is characterized in that: recombinant plasmid need be transformed in competence e. coli bl21 with expressed fusion protein.

3. the construction process of a recombinant plasmid claimed in claim 1, it is characterized in that: PCR method amplifies phycocyanin beta subunit gene, use BamH I, HindIII restriction endonuclease in 37 DEG C of double digestion 1.5h the gene fragment amplifying and plasmid vector pET-32a simultaneously, after agarose gel electrophoresis, cut glue and reclaim object product, 16 DEG C connect 16h.

4. according to claim 3, a kind of fusion rotein of engineering bacterium expression of structure, is characterized in that: fusion rotein, with after nickel affinity chromatography column purification, reaches electrophoresis pure.

5. fusion rotein according to claim 4, is characterized in that: the propagation of Hela cell is had to certain restraining effect.