CN101423842A - Method for synthesizing protein by using cultivated silkworm middle silk gland vitro expression system - Google Patents
Method for synthesizing protein by using cultivated silkworm middle silk gland vitro expression system Download PDFInfo
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Abstract
The invention discloses a method for synthesizing protein by using an in-vitro expression system of middle silk glands of silkworms. The method comprises the following steps: adding any one of pUC19-Serl-PolyA recombinant plasmids or pUC19-Serl-GFP-PolyA recombinant plasmids or recombinant plasmids inserted with exogenous genes into tissue extract of the middle silk glands of the silkworms; adding a protease inhibitor into the mixture; and treating the mixture in water bath at a temperature of between 25 and 29 DEG C, so as to obtain expresses of target protein. The method has the following advantages: the protein is expressed under a low temperature condition of between 25 and 29 DEG C, so the method is favorable for generating expression protein in a dissolving state and favorable for separation and purification of the protein. At the same time, the method is not limited by cells, and can synthesize the protein in a short time.
Description
Technical field
The present invention relates to a kind of protein synthetic method, this method has been utilized silkworm middle division of silkgland vivoexpression system.
Background technology
At present, extensively utilize gene recombination technology and use expression systems (hereinafter often abbreviating " cell system " as) such as yeast viable cell, insect cell as method for producing protein.But viable cell shows the tendency that exogenous protein disappears owing to its function keeps, and since the expression of cytotoxic protein matter in the viable cell has stoped cell to be grown exist many can not be by easy expressed protein.And the vivoexpression system does not have the restriction on the above-mentioned cell system protein synthesis comparatively speaking, and can be under the situation that does not injure organism synthetic protein.In addition because synthetic protein at short notice, thereby is compared in proteinic production without operations such as cultivations with cell system.In addition, because the vivoexpression system also is used for the protein that scale operation is made up of the aminoacid sequence that is not utilized by organism, thereby be expected to become promising expression method.
Silkworm (Bombyx mori) is a kind of powerful insect that secretes an ability that has.Fibroin by fibroin (fibroin, Fib) and silk gum (sericin Ser) forms.Middle division of silkgland is synthetic and the place of secretion silk gum, has the synthetic intensity of very high albumen, is rare in other zooblasts.Therefore, the sericin gene promoter is the natural promoter with strong space-time specificity and powerful startup ability.Existing at present 5 kinds of silk gum genes are cloned and are separated, and are called as Ser1, Ser2, MSGS3, MSCS4 and MSGS5 respectively.Wherein the Ser1 gene is the main expressing gene of sericin, and domestic silkworm silk glue protein gene ser1 has the advantages that to express in middle division of silkgland tissue specificity, high efficiency.Can utilize its promotor make up can expression alien gene the sericterium biological factory.At present, utilize silkworm middle division of silkgland tissue extract to carry out vivoexpression report is not also arranged
Summary of the invention
At the deficiencies in the prior art part; the invention provides a kind of silkworm middle division of silkgland vivoexpression system method for synthesizing protein that utilizes: in silkworm middle division of silkgland tissue extract, add pUC19-Ser1-PolyA recombinant plasmid or pUC19-Ser1-GFP-PolyA recombinant plasmid or be inserted with above any recombinant plasmid of foreign gene; add proteinase inhibitor; 25 ℃～29 ℃ water-baths, the expression that promptly gets target protein.
The preparation method of described silkworm middle division of silkgland tissue extract comprises:
(1) get four ages or five age silkworm, silkworm is dipped in 75% ethanol anaesthetizes, dissect and take out sericterium, place physiological saline or the PBS of 0.9 ℅, get middle division of silkgland ,-80～-60 ℃ of preservations;
(2) get the middle division of silkgland of above preservation, place homogenizer, add physiological saline or PBS, homogenate on ice; Get homogenate in 1.5ml EP pipe, 2 ℃～6 ℃, the centrifugal 10～15min of 10000～14000rpm gets supernatant, and triplicate is got supernatant, is silkworm middle division of silkgland tissue extract.
Described pUC19-Ser1-PolyA construction of recombinant plasmid, its feature may further comprise the steps:
(1) be template with domestic silkworm gene group DNA, L-1 and L-2 are primer, and pcr amplification goes out the Ser1 promoter sequence, and directed cloning obtains recombinant plasmid pUC19-Ser1 to carrier pUC19 behind EcoR I and BamHI double digestion;
L-1：5′-ccggaattcgaaattcttagctacatct-3′；
L-2：5′-ggcggatccccgggtaccgagctcgttggcggtctttggatc-3′；
(2) be template with domestic silkworm gene group DNA, L-3 and L-4 are primer, and pcr amplification goes out Ser1 polyA (1), and directed cloning pUC19-Ser1 promotor downstream to the pUC19-FL obtains recombinant plasmid pUC19-Ser1-PolyA behind SalI and HindIII double digestion;
L-3：5′-gcggtcgacctgcaggcatgcggttccagcacaagtggaggag-3′；
L-4：5′-gccaagcttgataatacgtttattatct-3′。
Described pUC19-Ser1-GFP-PolyA construction of recombinant plasmid, its feature may further comprise the steps:
(1) obtains recombinant plasmid pUC19-Ser1 by the described step of claim 3 (1);
(2) being template with domestic silkworm gene group DNA, is primer with L-5 and L-6, and pcr amplification goes out Ser1 polyA (2);
L-5：5′-gagctgtacaagtaaggttccagcacaagtggaggag-3′；
L-6：5′-gccaagcttgataatacgtttattatct-3′；
(3), be template with plasmid pEGFP-N1, be primer with L-7 and L-8, pcr amplification GFP reporter gene sequence;
L-7：5′-ccggtcgacctgcaggcatgcatggtgagcaagggc-3′；
L-8：5′-cacttgtgctggaaccttacttgtacagctc-3′；
(4) be template with Ser1 polyA (2) and GFP reporter gene sequence, with L-7 and L-6 is primer, utilize the method for overlapping PCR to amplify the fragment that has Ser1 polyA (2) and GFP reporter gene sequence, be GFP-PolyA, directed cloning obtains recombinant plasmid pUC19-Ser1-GFP-PolyA to recombinant plasmid pUC19-Ser1 behind SalI and HindIII double digestion.
Usefulness of the present invention is: expressing protein under 25 ℃～29 ℃ cold condition, and help expressing protein and generate with dissolved state, be beneficial to proteic separation and purification.Simultaneously, method of the present invention is not subjected to the restriction of cell, synthetic protein at short notice.
Description of drawings
The recombinant plasmid pUC19-Ser1-PolyA of Fig. 1, embodiments of the invention;
The recombinant plasmid pUC19-Ser1-GFP-PolyA of Fig. 2, embodiments of the invention;
The electrophorogram of the detection protein expression of Fig. 3, embodiments of the invention;
Among the figure: M, standard molecular weight albumen; Sampling in 1,0 hour; Sampling in 2,3 hours; Sampling in 3,6 hours; Sampling in 4,12 hours; Sampling in 5,18 hours.
Embodiment
The present invention chooses the silkworm in four ages or five periods in age, and dissection places homogenizer after obtaining the silkworm middle division of silkgland, adds an amount of physiological saline or PBS, homogenate on ice, and centrifugal acquisition supernatant liquor is silkworm middle division of silkgland extracting solution.Silkworm middle division of silkgland extracting solution is hatched in 25 ℃～29 ℃ water-baths, divide 0,3h, 6h, 12h, 18h, the 24h sampling identifies with the SDS-PAGE gel electrophoresis whether this system has the ability of synthetic proteins.
Construction of recombinant plasmid:
1, recombinant plasmid pUC19-Ser1-PolyA
With domestic silkworm gene group DNA (Genbank M76430) is template, L-1 and L-2 are primer, pcr amplification goes out Ser1 promoter sequence (about 1656bp), and directed cloning obtains recombinant plasmid pUC19-Ser1 to carrier pUC19 (promega company) behind EcoRI and BamHI double digestion.Be template with domestic silkworm gene group DNA (Genbank M76430) equally, L-3 and L-4 are primer, pcr amplification goes out the polyA tailing signal (about 593bp) of Ser1, directed cloning Ser1 promotor downstream to the pUC19-Ser1 obtains recombinant plasmid pUC19-Ser1-PolyA (Fig. 1) behind SalI and HindIII double digestion.
2, recombinant plasmid pUC19-Ser1-GFP-PolyA
With domestic silkworm gene group DNA (Genbank M76430) is template, is primer with L-5 and L-6, and pcr amplification goes out the polyA tailing signal sequence (about 593bp) of Ser1, i.e. Ser1 polyA (2); With plasmid pEGFP-N1 (Genbank U55762) is template, is primer with L-7 and L-8, pcr amplification GFP reporter gene sequence (about 720bp).Be template with Ser1 polyA (2) and GFP reporter gene sequence (GenbankNM_001044041 and Genbank U55762) subsequently, with L-7 and L-6 is primer, utilize the method for overlapping PCR to amplify the fusion sequence GFP-PolyA that has Ser1 polyA (2) and GFP reporter gene sequence, GFP-PolyA directed cloning behind SalI and HindIII double digestion obtains recombinant plasmid pUC19-Ser1-GFP-PolyA (Fig. 2) to recombinant plasmid pUC19-Ser1.
Utilize silkworm middle division of silkgland vivoexpression system synthetic proteins.Reaction system: in silkworm middle division of silkgland tissue extract, add pUC19-Ser1-PolyA recombinant plasmid or pUC19-Ser1-GFP-PolyA recombinant plasmid or be inserted with the pUC19-Ser1-GFP-PolyA recombinant plasmid or the pUC19-Ser1-PolyA recombinant plasmid of foreign gene; add proteinase inhibitor; 25 ℃～29 ℃ water-baths; divide 0; 3h, 6h, 12h; 18h, the 24h sampling.Utilize native polyacrylamide gel electrophoresis to detect protein expression.
Embodiment
One, the preparation of domestic natural silk gland
Get four ages or five age silkworm, silkworm is dipped in 75% ethanol anesthesia takes out after 30 seconds, fixedly silkworm head and afterbody are on cured dish with pin, the outside of belly is cut off its belly with the dissection scissors up, launching about skin, fixes with pin.Take the photograph pincers with dissection and live rectum, one mentions towards cephalad direction, and one side is cut off the tracheae and the muscle of climbing.After removing alimentary canal, like this about two ones sericterium just exposed, with tweezers sericterium is taken out, place physiological saline or the PBS of 0.9 ℅.With scissors front middle part sericterium and posterior division of silkgland are separated subsequently.-70 ℃ of preservations are standby.
Two, the preparation of silkworm middle division of silkgland extracting solution
Get the middle division of silkgland of 10 silkworms, place homogenizer, add 1ml physiological saline or PBS, homogenate on ice; Draw homogenate in 1.5ml EP pipe, 4 ℃, the centrifugal 10min of 12000rpm gets supernatant, triplicate; Draw supernatant to new 1.5ml EP pipe, with the packing of 500ul pipe, every pipe 50ul is silkworm middle division of silkgland extracting solution, promptly with or deposit in the liquid nitrogen.
Three, the extraction of silkworm middle division of silkgland genomic dna
Take out middle division of silkgland from 85 ages the children silkworm, add liquid nitrogen and smash to pieces, add silkworm middle division of silkgland extracting solution (the 10mmol/L Tris-HCl (pH8.0) of the above-mentioned gained of 2mL, 0.1mmol/L EDTA (pH8.0), 1%SDS, 500mmol/L NaCl), add Proteinase K (promega) (making final concentration reach 250ug/mL) behind the mixing, 50 ℃ leave standstill 12～24h, add isopyknic phenol, put upside down 10min lentamente back and forth, centrifugal, get supernatant liquor, add RNA enzyme (sigma), leave standstill 30min in 37 ℃; Successively add isopyknic phenol: chloroform (1:1) and chloroform: each extracting of primary isoamyl alcohol 1 time, get the dehydrated alcohol deposit D NA that supernatant liquor adds 2 times of volumes, carefully twine out DNA with thin glass stick, 70% ethanol is washed 1 time, after 37 ℃ of dryings, is dissolved in the TE damping fluid.
Four, construction of recombinant plasmid
With reference to the silkworm sericin-1 gene 5 ' end upstream sequence of having announced among the GenBank (GenbankAB007831), a pair of primer that synthetic cloning promoter sequence is used:
L-1:5 '-ccg
GaattcGaaattcttagctacatct-3 ', underscore represent EcoR I site;
L-2:5 '-ggc
GgatccCcgggtaccgagctcgttggcggtctttggatc-3 ', underscore represent the BamHI site;
Two pairs of PCR primers using with reference to the polyA tailing signal sequence of the synthetic clone of the silkworm sericin-1 sequence of having announced among the GenBank (Genbank NM_001044041) Ser1 gene:
L-3:5 '-gcg
GtcgacCtgcaggcatgcggttccagcacaagtggaggag-3 ', underscore represent the SalI site;
L-4:5 '-gcc
AagcttGataatacgtttattatct-3 ', underscore represent the HindIII site;
L-5：5′-gagctgtacaagtaaggttccagcacaagtggaggag-3′；
L-6:5 '-gcc
AagcttGataatacgtttattatct-3 ', underscore represent the HindIII site);
With reference to plasmid pEGFP-N1 sequence, synthetic clone's green fluorescent protein (Green FluorescentProtein, GFP) two pairs of PCR primers using of reporter gene:
L-7:5 '-ccgg
TcgacCtgcaggcatgcatggtgagcaagggc-3 ', underscore represent the SalI site;
L-8：5′-cacttgtgctggaaccttacttgtacagctc-3′。Primer synthesizes and the order-checking of PCR product is finished by the living worker in Shanghai bio-engineering corporation.
1.ser1 the amplification of promoter sequence
With domestic silkworm gene group DNA (Genbank M76430) is template, and the PCR reaction parameter is 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCR?Buffer 10μl
25mmol?MgCl2 8μl
2.5mmol?dNTPs 8μl
L-1 1μl
L-2 1μl
KOD-Plus archaeal dna polymerase (TOYOBO company, Japan) 1 μ l
Add aseptic double-distilled water to 100 μ l
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments, cut glue simultaneously and reclaimed the purpose fragment, obtained the ser1 promotor.
2.ser1 the amplification of polyA tailing signal sequence (1)
With domestic silkworm gene group DNA (Genbank M76430) is template, and the PCR reaction parameter is 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCR?Buffer 10μl
25mmol?MgCl 28μl
2.5mmol?dNTPs 8μl
L-3 1μl
L-4 1μl
KOD-Plus archaeal dna polymerase (TOYOBO company, Japan) 1 μ l
Add aseptic double-distilled water to 100 μ l
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments, cut glue simultaneously and reclaimed the purpose fragment, obtained ser1 polyA (1).
3.ser1 the amplification of polyA tailing signal sequence (2)
With domestic silkworm gene group DNA (Genbank M76430) is template, and the PCR reaction parameter is 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCR?Buffer 10μl
25mmol?MgCl2 8μl
2.5mmol?dNTPs 8μl
L-5 1μl
L-6 1μl
KOD-Plus archaeal dna polymerase (TOYOBO company, Japan) 1 μ l
Add aseptic double-distilled water to 100 μ l
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments, cut glue simultaneously and reclaimed the purpose fragment, obtained ser1polyA (2).This purpose fragment can be used as template Ser1-polyA and is used for subsequent experimental.
4.GFP the amplification of reporter gene sequence
With plasmid pEGFP-N1 (GE company, Genbank U55762) is template, and the PCR reaction parameter is 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCR?Buffer 10μl
25mmol?MgCl 28μl
2.5mmol?dNTPs 8μl
L-7 1μl
L-8 1μl
KOD-Plus archaeal dna polymerase (TOYOBO company, Japan) 1 μ l
Add aseptic double-distilled water to 100 μ l
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments, cut glue simultaneously and reclaimed the purpose fragment.This purpose fragment can be used as template GFP and is used for subsequent experimental.
5.ser1 polyA tailing signal sequence (2) and the fusion of GFP reporter gene sequence
The PCR reaction parameter is 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCR?Buffer 10μl
25mmol?MgCl2 8μl
2.5mmol?dNTPs 8μl
L-7 1μl
L-6 1μl
Template Ser1-polyA (2) 1 μ l
Template GFP (available from preceding step) 1 μ l
KOD-Plus archaeal dna polymerase (TOYOBO company, Japan) 1 μ l
Add aseptic double-distilled water to 100 μ l
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments.
Because added the partial sequence of the polyA tailing signal sequence 5 ' end of ser1 during the downstream primer L-8 of GFP reporter gene design, so the polyA tailing signal sequence 5 ' terminal sequence of 3 ' terminal sequence of the GFP reporter gene that pcr amplification goes out and ser1 exists overlapping, the downstream primer S-5 of the polyA tailing signal sequence of ser1 has added the partial sequence of GFP reporter gene 5 ' end when designing, so 3 ' terminal sequence of the polyA tailing signal sequence of the ser1 that pcr amplification goes out and 5 ' terminal sequence of GFP reporter gene exist overlapping, utilize PCR method just can amplify the polyA tailing signal sequence of ser1 and the fusion sequence of GFP reporter gene, make up GFP-PolyA.
6. the structure of recombinant plasmid pUC19-Ser1
The Ser1 promoter sequence fragment that pcr amplification in the step 1 is gone out is connected on the plasmid pUC19 (TAKARA, precious biological).Earlier respectively Ser1 promoter sequence fragment and plasmid pUC19 are carried out EcoRI, BamHI double digestion, 37 ℃ of reactions are reclaimed enzyme respectively after 2 hours and are cut product, connect again.
In an aseptic 0.2ml centrifuge tube, mix following ingredients:
Ser1 promoter sequence fragment 2 μ l
Plasmid pUC19 0.5 μ l
2×Rapid?Ligation?buffer 5μl
T4 DNA ligase (available from promage company) 1 μ l
Add aseptic double-distilled water to 10 μ l, mixing.Behind 25 ℃ of reaction 1h, reaction mixture is converted among the competent cell E.coli DH5 α.Picking list bacterium colony extracts plasmid, carries out enzyme and cuts preliminary evaluation, and through order-checking, sequence is entirely true.
7. the structure of recombinant plasmid pUC19-Ser1-PolyA
Respectively Ser1 polyA (1) and plasmid pUC19-Ser1 are carried out SaII, HindIII double digestion, 37 ℃ of reactions are reclaimed enzyme respectively after 2 hours and are cut product, connect again.
The polyA tailing signal sequence (1) that pcr amplification is gone out is connected on the plasmid pUC19-Ser1.
In an aseptic 0.2ml centrifuge tube, mix following ingredients:
Ser1?polyA(1) 2μl
Plasmid pUC19-Ser1 0.5 μ l
2×Rapid?Ligation?buffer 5μl
T4 DNA ligase (available from promage company) 1 μ l
Add aseptic double-distilled water to 10 μ l, mixing.Behind 25 ℃ of reaction 1h, reaction mixture is converted among the competent cell E.coli DH5 α.Picking list bacterium colony extracts plasmid, carries out enzyme and cuts preliminary evaluation, and through order-checking, sequence is entirely true.
8. the structure of recombinant plasmid pUC19-Ser1-GFP-PolyA
The fusion fragment GFP-PolyA that pcr amplification is gone out is connected on the plasmid pUC19-Ser1.Respectively plasmid pUC19-Ser1 and fusion fragment GFP-PolyA are carried out SaII, HindIII double digestion, 37 ℃ of reactions are reclaimed enzyme respectively after 2 hours and are cut product, connect again.
In an aseptic 0.2ml centrifuge tube, mix following ingredients:
Merge fragment GFP-PolyA 2 μ l
Plasmid pUC19-Ser1 0.5 μ l
2×Rapid?Ligation?buffer 5μl
T4DNA ligase (available from promage company) 1 μ l
Add aseptic double-distilled water to 10 μ l, mixing.Behind 25 ℃ of reaction 1h, reaction mixture is converted among the competent cell E.coli DH5 α.Picking list bacterium colony extracts plasmid, carries out enzyme and cuts preliminary evaluation, and through order-checking, sequence is entirely true.
9, foreign gene inserts carrier pUC19-Ser1-PolyA or pUC19-Ser1-GFP-PolyA
Goal gene is increased out, add restriction enzyme site (optional two of BamHI, XbaI, SaII three) simultaneously at the two ends of goal gene, simultaneously goal gene and carrier (pUC19-Ser1-PolyA or pUC19-Ser1-GFP-PolyA) are carried out double digestion again, reclaim enzyme and cut product, under the effect of T4 ligase enzyme, goal gene can be connected in the carrier.Operating method is with above-mentioned method.
Five, utilize silkworm middle division of silkgland vivoexpression system method for synthesizing protein
In the centrifuge tube that 50ul silkworm middle division of silkgland tissue extract is housed, add about 1ng pUC19-Ser1-GFP-PolyA recombinant plasmid or pUC19-Ser1-PolyA recombinant plasmid (or being inserted with the pUC19-Ser1-GFP-PolyA recombinant plasmid of foreign gene or the pUC19-Ser1-PolyA recombinant plasmid of insertion foreign gene) and proteinase inhibitor; 25 ℃～29 ℃ water-baths; divide 0; 3h; 6h; 12h, 18h, the 24h sampling detects proteic expression.See Fig. 3.
Six, the detection of expression product
Electrophoretic method (seeing " molecular cloning ") detects proteic expression.If used carrier is pUC19-Ser1-GFP-PolyA or the pUC19-Ser1-GFP-PolyA recombinant plasmid that is inserted with foreign gene, after then electrophoresis finishes, under fluorescent microscope, observe, under the 488nm excitation wavelength, observe the expression of green fluorescent protein GFP.If used carrier is the pUC19-Ser1-PolyA recombinant plasmid that has the external source goal gene, then adopts and examine the expression of dying method observation target protein.
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ?ID?NO?1：
SEQ?ID?NO?2：
SEQ?ID?NO?3：
SEQ?ID?NO?4：
SEQ?ID?NO?5：
SEQ?ID?NO?6：
SEQ?ID?NO?7：
SEQ?ID?NO?8：
Claims (4)
1, a kind of silkworm middle division of silkgland vivoexpression system method for synthesizing protein that utilizes; it is characterized in that: in silkworm middle division of silkgland tissue extract, add pUC19-Ser1-PolyA recombinant plasmid or pUC19-Ser1-GFP-PolyA recombinant plasmid or be inserted with above any recombinant plasmid of foreign gene; add proteinase inhibitor; 25 ℃～29 ℃ water-baths, the expression that promptly gets target protein.
2, the silkworm middle division of silkgland vivoexpression system method for synthesizing protein that utilizes according to claim 1 is characterized in that the preparation method of described silkworm middle division of silkgland tissue extract comprises:
(1) get four ages or five age silkworm, silkworm is dipped in 75% ethanol anaesthetizes, dissect and take out sericterium, place physiological saline or the PBS of 0.9 ℅, get middle division of silkgland ,-80～-60 ℃ of preservations;
(2) get the middle division of silkgland of above preservation, place homogenizer, add physiological saline or PBS, homogenate on ice; Get homogenate in 1.5ml EP pipe, 2 ℃～6 ℃, the centrifugal 10～15min of 10000～14000rpm gets supernatant, and triplicate is got supernatant, is silkworm middle division of silkgland tissue extract.
3, the silkworm middle division of silkgland vivoexpression system method for synthesizing protein that utilizes according to claim 1 is characterized in that described pUC19-Ser1-PolyA construction of recombinant plasmid, and its feature may further comprise the steps:
(1) be template with domestic silkworm gene group DNA, L-1 and L-2 are primer, and pcr amplification goes out the Ser1 promoter sequence, and directed cloning obtains recombinant plasmid pUC19-Ser1 to carrier pUC19 behind EcoR I and BamHI double digestion;
L-1：5′-ccggaattcgaaattcttagctacatct-3′；
L-2：5′-ggcggatccccgggtaccgagctcgttggcggtctttggatc-3′；
(2) be template with domestic silkworm gene group DNA, L-3 and L-4 are primer, and pcr amplification goes out Ser1 polyA (1), and directed cloning pUC19-Ser1 promotor downstream to the pUC19-FL obtains recombinant plasmid pUC19-Ser1-PolyA behind SalI and HindIII double digestion;
L-3：5′-gcggtcgacctgcaggcatgcggttccagcacaagtggaggag-3′；
L-4：5′-gccaagcttgataatacgtttattatct-3′。
4, the silkworm middle division of silkgland vivoexpression system method for synthesizing protein that utilizes according to claim 1 is characterized in that described pUC19-Ser1-GFP-PolyA construction of recombinant plasmid, and its feature may further comprise the steps:
(1) obtains recombinant plasmid pUC19-Ser1 by the described step of claim 3 (1);
(2) being template with domestic silkworm gene group DNA, is primer with L-5 and L-6, and pcr amplification goes out Ser1 polyA (2);
L-5：5′-gagctgtacaagtaaggttccagcacaagtggaggag-3′；
L-6：5′-gccaagcttgataatacgtttattatct-3′；
(3), be template with plasmid pEGFP-N1, be primer with L-7 and L-8, pcr amplification GFP reporter gene sequence;
L-7：5′-ccggtcgacctgcaggcatgcatggtgagcaagggc-3′；
L-8：5′-cacttgtgctggaaccttacttgtacagctc-3′；
(4) be template with Ser1 polyA (2) and GFP reporter gene sequence, with L-7 and L-6 is primer, utilize the method for overlapping PCR to amplify the fragment that has Ser1polyA (2) and GFP reporter gene sequence, be GFP-PolyA, directed cloning obtains recombinant plasmid pUC19-Ser1-GFP-PolyA to recombinant plasmid pUC19-Ser1 behind SalI and HindIII double digestion.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226202A (en) * | 2011-05-13 | 2011-10-26 | 浙江大学 | Method for synthetizing secretion lysozyme by middle silkgland cell of silkworm |
| CN102242147A (en) * | 2011-05-13 | 2011-11-16 | 浙江同点生物科技有限公司 | Method for synthesizing and secreting rabies virus nucleoprotein in middle silkworm silk-gland cells |
| CN102286529A (en) * | 2011-05-13 | 2011-12-21 | 浙江大学 | Method for synthesizing silkworm silk gland cells capable of secreting rabies virus glucoprotein |
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2008
- 2008-08-29 CN CNA2008101204706A patent/CN101423842A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226202A (en) * | 2011-05-13 | 2011-10-26 | 浙江大学 | Method for synthetizing secretion lysozyme by middle silkgland cell of silkworm |
| CN102242147A (en) * | 2011-05-13 | 2011-11-16 | 浙江同点生物科技有限公司 | Method for synthesizing and secreting rabies virus nucleoprotein in middle silkworm silk-gland cells |
| CN102286529A (en) * | 2011-05-13 | 2011-12-21 | 浙江大学 | Method for synthesizing silkworm silk gland cells capable of secreting rabies virus glucoprotein |
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