CN104073518A - Method for preparing recombinant human epidermal growth factor by adopting castor silkworm chrysalis as bioreactor - Google Patents
Method for preparing recombinant human epidermal growth factor by adopting castor silkworm chrysalis as bioreactor Download PDFInfo
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- CN104073518A CN104073518A CN201410160406.6A CN201410160406A CN104073518A CN 104073518 A CN104073518 A CN 104073518A CN 201410160406 A CN201410160406 A CN 201410160406A CN 104073518 A CN104073518 A CN 104073518A
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Abstract
The invention discloses a method for preparing a recombinant human epidermal growth factor by adopting castor silkworm chrysalis as a bioreactor, and belongs to the field of genetic engineering. A started vector pENTR<TM>4Dual-6xHis-HCEGF is integrated to the downstream of a destination vector linearized AnpeNPVDNA nuclear polygonal body gene promoter by a genetic engineering technology; a recombinant baculovirus 6xHis-HCEGF-AnpeNPVDNA of an expression vector is obtained; the expression vector is inoculated with castor silkworm chrysalis after being processed; the HCEGF is efficiently expressed; the recombinant human epidermal growth factor with the efficacy similar to that of a natural product is prepared by incubation, separation and purification. The method belongs to an eukaryotic cell gene expression method, and has a protein post-translational modification process and high expression efficiency, the biological characteristics of the expression product are similar to those of the natural product, and the silkworm chrysalis material is abundant in source, and has important significance in improvement of the economic value of the castor silkworm and promotion of sericulture career development in Guangxi.
Description
Technical field
The present invention relates to genetically engineered field, be specifically related to that castor-oil plant silkworm chrysalis makees bio-reactor, recombinant baculovirus does the method that expression vector is prepared recombinant human epidermal growth factor.
Background technology
The polypeptide that human epidermal growth factor (human epidermal growth factor, hEGF) is made up of 53 amino acid, molecular weight is about 6000Da, and its biological function makes it have a wide range of applications in pharmacy and cosmetic field.Human epidermal growth factor has the function that promotes epithelium regeneration, can be for promoting the healing of surgical incision and other surface of a wound, as the scratch of dermatoplasty, skin, burn, erosion etc.; Also there is the effect of the propagation that promotes corneal epithelial cell, be used for treating corneal injury, ulcer and promoting the survival of corneal transplant; Can also gastric acid secretion inhibiting, can be used for the treatment of peptide ulceration; Can also accelerate epidermal renewal, make the rejuvenation of epidermic cell Individual Age, play the cosmetic results such as skin care, crease-resistant, whitening, can be used for cosmetic field.Thereby, obtain similar natural recombinant human epidermal growth factor and there is important economic worth.
Human epidermal growth factor once successfully expresses in intestinal bacteria, subtilis, yeast and silkworm baculovirus, does method that expression vector prepares recombinant human epidermal growth factor there are no report but utilize castor-oil plant silkworm chrysalis to do bio-reactor, recombinant baculovirus.Castor-oil plant silkworm chrysalis does the method that bio-reactor, recombinant baculovirus do expression vector and belongs to system of gene expression in eukaryote method, has protein post-translational modification process, the advantage such as expression efficiency is high, and the biological characteristics of expression product is similar to natural product.
Guangxi is unique Semen Ricini silkworm conservation base, has ripe rearing of eri-silkworm technology and abundant cassava leaves feed resource.Utilize castor-oil plant silkworm cocoon processing silk silk floss, silkworm chrysalis to do the new purposes of bio-reactor, to promoting the economic worth of Semen Ricini silkworm, promote that the sericulture career development in Guangxi is significant.Silkworm chrysalis used has cheapness, can store, and convenient transportation, need not raise or cell cultures, can mechanized operation etc. advantage.
Summary of the invention
Goal of the invention of the present invention is: provide a kind of with castor-oil plant silkworm chrysalis make bio-reactor, recombinant baculovirus does the method that expression vector is prepared recombinant human epidermal growth factor.
For achieving the above object, the technical solution used in the present invention is as follows: by genetic engineering technique by entry vector pENTR
tM4Dual-6xHis-HCEGF is incorporated into object carrier linearizing AnpeNPV DNA core polyhedrosis gene promotor downstream, obtain expression vector restructuring tussah baculovirus 6xHis-HCEGF-AnpeNPV DNA, described expression vector is inoculated castor-oil plant silkworm chrysalis after treatment, high efficient expression HCEGF, through hatching, the similar natural product of effect is prepared in separation and purification recombinant human epidermal growth factor; Wherein:
(1) construction process of described entry vector is:
With Nco I/EcoR I double digestion digestion HCEGF/pET-28a plasmid, acquisition has the entire reading frame frame (orf) and the N that finish to terminator codon TAA from initiator codon ATG and holds the 6xHis-HCEGF gene fragment with 6xHis, and this gene fragment is inserted into the pENTR that Nco I/EcoR I double digestion is crossed
tMbetween the Nco I and EcoR I of 4Dual plasmid, obtain entry vector pENTR
tM4Dual-6xHis-HCEGF, makes between the attL1 and attL2 of 6xHis-HCEGF fragment in plasmid;
(2) construction process of described object carrier is:
Extract tussah baculovirus AnpeNPV DNA, by DNA homology on the same group technology insert by the connected attR1 of Avr II restriction enzyme site and aatR2 sequence construct object carrier A npeNPV in the core polyhedrosis gene promotor downstream of AnpeNPV DNA, extracting AnpeNPV DNA by Avr II linearization for enzyme restriction;
(3) construction process of described expression vector is:
Get described entry vector pENTR
tM4Dual-6xHis-HCEGF150ng, object carrier linearizing AnpeNPVDNA150ng, 5 × LR clone enzyme mixture 2 μ l, pH8.0TB damping fluid are mixed to 10 μ l, and 25 DEG C of temperature are bathed 1-16 hour; PENTR
tM4Dual-6xHis-HCEGFDNA and linearizing AnpeNPV DNA recombinate under the effect of LR clone enzyme, generate expression vector restructuring tussah baculovirus 6xHis-HCEGF-AnpeNPV DNA.
(2) treatment process before described expression vector inoculation castor-oil plant silkworm chrysalis is:
With described restructuring tussah baculovirus 6xHis-HCEGF-AnpeNPV DNA inoculation tussah host AnPe cell, cultivate for 27 DEG C and after 1 week, collect supernatant expression vector virus liquid and repeatedly carry out spot formation method after enlarged culturing and measure infectivity PFU and-80 DEG C and save backup.
Further, described in the temperature of hatching be 27 DEG C, incubation time is 1 week.
Further, first described separation and purification is that the silkworm chrysalis after hatching is ground, then collect supernatant liquor, then carry out ammonium sulfate precipitation and the separation and purification of Ni-NTA Agrose affinity chromatography, finally obtain the similar natural product of effect recombinant human epidermal growth factor.
In sum, owing to having adopted technique scheme, the invention has the beneficial effects as follows:
(1) the present invention utilizes castor-oil plant silkworm chrysalis to make bio-reactor, recombinant baculovirus does expression vector and prepares recombinant human epidermal growth factor, belong to system of gene expression in eukaryote method, there is protein post-translational modification process, expression efficiency is high, the advantage that the biological characteristics of expression product is similar to natural product.
(2) Guangxi is unique Semen Ricini silkworm conservation base, has ripe rearing of eri-silkworm technology and abundant cassava leaves feed resource, and therefore, silkworm chrysalis material source is abundant.Castor-oil plant silkworm chrysalis has cheapness, easily stores, convenient transportation, need not raising or cell cultures, can mechanized operation etc. advantage.Residue cocoon shell also can be processed the comprehensive utilizations such as silk cotton, thereby, to promoting the economic worth of Semen Ricini silkworm, promote that the sericulture career development in Guangxi is significant.
Brief description of the drawings
Fig. 1: entry vector 6xHis-HCEGF structural representation.
Fig. 2: restructuring tussah baculovirus 6xHis-HCEGF-AnpeNPV builds schematic diagram.
Fig. 3: the gene of recombinant human epidermal growth factor and aminoacid sequence figure (6xHis-HCEGF).
Embodiment
Below by following examples, the invention will be further described.
Embodiment 1 builds entry vector
With Nco I/EcoR I double digestion digestion HCEGF/pET-28a plasmid (University Of Suzhou, patent No. CN1644691A), acquisition has the 6xHis-HCEGF gene fragment that finishes entire reading frame frame (orf) and the subsidiary 6xHis of N end from initiator codon ATG to terminator codon TAA, and this gene fragment is inserted into the pENTR that Nco I/EcoR I double digestion is crossed
tMbetween the Nco I and EcoR I of 4Dual plasmid (Invitrogen company), obtain entry vector pENTR
tM4Dual-6xHis-HCEGF, makes between the attL1 and attL2 of 6xHis-HCEGF fragment in plasmid.
Embodiment 2 builds object carrier
Extract tussah baculovirus AnpeNPV DNA, by DNA homology on the same group technology insert by the connected attR1 of Avr II restriction enzyme site and aatR2 sequence construct object carrier A npeNPV in the core polyhedrosis gene promotor downstream of AnpeNPV DNA, extracting AnpeNPVDNA by Avr II linearization for enzyme restriction.
Embodiment 3 construction of expression vector
Get entry vector pENTR
tM4Dual-6xHis-HCEGF150ng, object carrier linearizing AnpeNPV DNA150ng, 5 × LR clone enzyme mixture 2 μ l, pH8.0TB damping fluid are mixed to 10 μ l, and 25 DEG C of temperature are bathed 1-16 hour.PENTR
tM4Dual-6xHis-HCEGFDNA and linearizing AnpeNPV DNA recombinate under the effect of LR clone enzyme, generate expression vector restructuring tussah baculovirus 6xHis-HCEGF-AnpeNPV DNA.
Processing before embodiment 4 expression vector inoculation castor-oil plant silkworm chrysalises
With described restructuring tussah baculovirus 6xHis-HCEGF-AnpeNPVDNA inoculation tussah host AnPe cell, cultivate for 27 DEG C and after 1 week, collect supernatant expression vector virus liquid and repeatedly carry out spot formation method after enlarged culturing and measure infectivity PFU and-80 DEG C and save backup.
Embodiment 5 expression vector inoculation castor-oil plant silkworm chrysalises and sample modulation
With 5 × 10
5(the healthy castor-oil plant silkworm chrysalis of 50 μ 6xHis-HCEGF-AnpeNPV virus liquid injection inoculation l), puts 27 DEG C of incubations to PFU.To inoculate the same day as the 0th day, after testing, in postvaccinal the 6th day (hatching after 1 week) silkworm chrysalis, recombinant human epidermal growth factor expression amount higher (0.2 μ g/g silkworm chrysalis) and silkworm chrysalis are without liquefaction, therefore, collect the inoculation silkworm chrysalis of latter the 6th day, after weighing, add the PBS damping fluid of pH6.8 and the benzene thiocarbamide of 1 μ g/g to carry out homogenate grinding by 0.5ml/g.The centrifugal 5min of 1700 × g, gets supernatant as silkworm chrysalis extracting solution.
The separation and purification of embodiment 6 recombinant human epidermal growth factors
(1) remove heteroproteins: 1. in castor-oil plant silkworm chrysalis extracting solution, slowly add ammonium sulfate, to ammonium sulfate saturation ratio be 35%.2. adjust silkworm chrysalis liquid pH to 4.6 left and right with 1mol/L hydrochloric acid, 4 DEG C leave standstill 4～24h, utilize isoelectric point precipitation that target protein and heteroproteins are separated.3. 4 DEG C of 8000rpm, 30min is centrifugal, collecting precipitation.4. precipitation is heavily dissolved in 0.02mol/L PBS (pH7.4), 4 DEG C leave standstill 4～24h.5. dialysis method, except ammonium sulfate, is followed 0.45 μ m membrane filtration.
(2) Ni-NTA Agrose affinity chromatography separation and purification recombinant human epidermal growth factor: because the recombinant human epidermal growth factor of design contains 6 × His-label, therefore adopt the separation and purification of Ni-NTAAgrose affinity chromatography.1. binding buffer liquid (the 50mmol/L NaH of three times of column volumes
2pO
4, pH8.0,300mmol/L NaCl, 10mmol/L imidazoles) and balance resin, place stand-by.2. the castor-oil plant silkworm chrysalis extracting solution upper prop of having adjusted, adjusting flow velocity is 10 cylinders per hour.Lavation buffer solution (the 50mmol/L NaH of 10 column volumes
2p0
4, pH8.0,300mmol/L NaCI, 20mmol/L imidazoles) and wash post.3. the elution buffer of five column volumes (50mmol/L NaH
2pO
4, pH8.0,300mmol/L NaCI, 250mmol/L imidazoles) rinse, collect elutriant, in elutriant, be the target protein matter recombinant human epidermal growth factor of purifying.
(3) qualification of target protein: with the negative contrast of normal castor-oil plant silkworm chrysalis, Western blot analyzes the castor-oil plant silkworm chrysalis extracting solution after 6xHis-HCEGF-AnpeNPV virus infection, in result demonstration extracting solution, there is HCEGF protein specific immune response band, and in normal silkworm chrysalis there are no HCEGF specific immune response band, illustrate recombinant virus 6xHis-HCEGF-AnpeNPV in castor-oil plant silkworm chrysalis successful expression gene obtained HCEGF protein recombinant human epidermal growth factor.
Embodiment 7 detects the biologic activity of recombinant human epidermal growth factor:
(1) cell experiment
1. short cell enzyme activity: cultivate Balb/c3T3 l cell as test group with the recombinant human epidermal growth factor of embodiment 6 separation and purification, control group adopts standard protein (recombinant human epidermal growth factor of purchase) to cultivate Balb/c3T3 l cell.As shown in table 1 by mtt assay detection short cell enzyme activity result in 0.0625 μ g/L～1.0 μ g/L concentration range, as can be seen from the table, the recombinant human epidermal growth factor of the embodiment of the present invention 6 gained is the same with the standard protein of control group (recombinant human epidermal growth factor of purchase), there is enzymatic activity, the higher enzymatic activity of protein concentration is stronger, present dose-dependently, high reactivity concentration is 1.0 μ g/L.
Table 1 embodiment of the present invention 5 gained recombinant human epidermal growth factors and standard protein
Short cell enzyme activity result
2. proliferation: cultivate Balb/c3T3 cell as test group with the recombinant human epidermal growth factor of separation and purification, control group adopts standard protein (recombinant human epidermal growth factor of purchase) to cultivate Balb/c3T3 cell.The proliferation result that detects test group and control group through trypan blue method is as shown in table 2.Embodiment 6 gained recombinant human epidermal growth factors have short cell proliferation as can be seen from the table, under lower concentration, (1 μ g/L) is identical with the standard protein (recombinant human epidermal growth factor of purchase) of control group, in the time of 10 μ g/L, 100 μ g/L concentration, recombinant human epidermal growth factor is better than the standard protein (recombinant human epidermal growth factor of purchase) of control group
The proliferation of the recombinant human epidermal growth factor of table 2 different concns
| Group | 1（μg/L） | 10（μg/L） | 100（μg/L） |
| Test group | （4.231±0.456）×10 5 | （6.154±0.657）×10 5 | （7.528±1.237）×10 5 |
| Control group | （3.280±0.230）×10 5 | （3.290±0.670）×10 5 | （4.000±0.200）×10 5 |
2. experimentation on animals
(1) on Mouse Weight, the impact of sprouting tooth, opening eyes: the recombinant human epidermal growth factor subcutaneous injection SPF level kunming mice (1mg/kg) of separation and purification, result shows: 1. latter the 9th day beginning Mouse Weight of injection gathers way and be better than negative control group (physiological saline), and effect is identical with positive controls (recombinant human epidermal growth factor of purchase).2. to sprout tooth speed identical with positive controls (recombinant human epidermal growth factor of purchase) with time of opening eyes for newborn mice, is significantly better than negative control group.
Claims (3)
1. castor-oil plant silkworm chrysalis does the method that bio-reactor is prepared recombinant human epidermal growth factor, it is characterized in that: by genetic engineering technique by entry vector pENTR
tM4Dual-6xHis-HCEGF is incorporated into object carrier linearizing AnpeNPV DNA core polyhedrosis gene promotor downstream, obtain expression vector restructuring tussah baculovirus 6xHis-HCEGF-AnpeNPV DNA, described expression vector is inoculated castor-oil plant silkworm chrysalis after treatment, high efficient expression HCEGF, through hatching, the similar natural product of effect is prepared in separation and purification recombinant human epidermal growth factor; Wherein:
(1) construction process of described entry vector is:
With Nco I/EcoR I double digestion digestion HCEGF/pET-28a plasmid, acquisition has the entire reading frame frame (orf) and the N that finish to terminator codon TAA from initiator codon ATG and holds the 6xHis-HCEGF gene fragment with 6xHis, and this gene fragment is inserted into the pENTR that Nco I/EcoR I double digestion is crossed
tMbetween the Nco I and EcoR I of 4Dual plasmid, obtain entry vector pENTR
tM4Dual-6xHis-HCEGF, makes between the attL1 and attL2 of 6xHis-HCEGF fragment in plasmid;
(2) construction process of described object carrier is:
Extract tussah baculovirus AnpeNPV DNA, by DNA homology on the same group technology insert by the connected attR1 of Avr II restriction enzyme site and aatR2 sequence construct object carrier A npeNPV in the core polyhedrosis gene promotor downstream of AnpeNPV DNA, extracting AnpeNPV DNA by Avr II linearization for enzyme restriction;
(3) construction process of described expression vector is:
Get described entry vector pENTR
tM4Dual-6xHis-HCEGF150ng, object carrier linearizing AnpeNPVDNA150ng, 5 × LR clone enzyme mixture 2 μ l, pH8.0TB damping fluid are mixed to 10 μ l, and 25 DEG C of temperature are bathed 1-16 hour; PENTR
tM4Dual-6xHis-HCEGFDNA and linearizing AnpeNPV DNA recombinate under the effect of LR clone enzyme, generate expression vector restructuring tussah baculovirus 6xHis-HCEGF-AnpeNPVDNA;
(4) treatment process before described expression vector inoculation castor-oil plant silkworm chrysalis is: with described expression vector restructuring tussah baculovirus 6xHis-HCEGF-AnpeNPV DNA inoculation tussah host AnPe cell, cultivate for 27 DEG C and after 1 week, collect supernatant expression vector virus liquid and repeatedly carry out spot formation method after enlarged culturing and measure infectivity PFU and save backup with-80 DEG C.
2. castor-oil plant silkworm chrysalis according to claim 1 does the method that bio-reactor is prepared recombinant human epidermal growth factor, it is characterized in that: described in the temperature of hatching be 27 DEG C, incubation time is 1 week.
3. castor-oil plant silkworm chrysalis according to claim 2 does the method that bio-reactor is prepared recombinant human epidermal growth factor, it is characterized in that: first described separation and purification is that the silkworm chrysalis after hatching is ground, then collect supernatant liquor, carry out again ammonium sulfate precipitation and the separation and purification of Ni-NTA Agrose affinity chromatography, finally obtain the similar natural product of effect recombinant human epidermal growth factor.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1078260A (en) * | 1992-05-08 | 1993-11-10 | 辽宁省农业科学院大连生物技术研究所 | Produce the method for exogenous genes products with cocoon chrysalis |
| CN1405310A (en) * | 2001-08-15 | 2003-03-26 | 浙江中奇生物药业股份有限公司 | Method for producing medicine using silkworm expressed numan epidermal growth factor |
| CN1644691A (en) * | 2004-12-21 | 2005-07-27 | 苏州大学 | Human epidermis factor nucleic sequence and use thereof |
-
2014
- 2014-04-21 CN CN201410160406.6A patent/CN104073518A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1078260A (en) * | 1992-05-08 | 1993-11-10 | 辽宁省农业科学院大连生物技术研究所 | Produce the method for exogenous genes products with cocoon chrysalis |
| CN1405310A (en) * | 2001-08-15 | 2003-03-26 | 浙江中奇生物药业股份有限公司 | Method for producing medicine using silkworm expressed numan epidermal growth factor |
| CN1644691A (en) * | 2004-12-21 | 2005-07-27 | 苏州大学 | Human epidermis factor nucleic sequence and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 伍玉婷 等: ""细胞实验检测蓖麻蚕蛹生物反应器表达的重组人表皮生长因子的生物学活性"", 《广西医学》, vol. 34, no. 6, 30 June 2012 (2012-06-30) * |
| 伍玉婷 等: ""蓖麻蚕蛹生物反应器生产重组人表皮生长因子的研究"", 《生物医学工程学杂志》, vol. 30, no. 1, 28 February 2013 (2013-02-28) * |
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