CN102978239A - Method for constructing two novel prawn expression vectors - Google Patents
Method for constructing two novel prawn expression vectors Download PDFInfo
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Abstract
The invention discloses a method for constructing two novel prawn expression vectors. A gene-specific primer is designed, a prawn white spot syndrome virus (WSSV) virus is taken as a template, different length fragments of immediately-early gene (IE) (-94/+52) and IE (-945/+52) of an immediately-early gene promoter IE 1 are amplified; and a restriction enzyme digestion and connection method is utilized, the IE (-94/+52) and the IE (-945/+52) are taken to replace a cytomegalovirus (CMV) promoter of an eukaryotic expression vector pc deoxyribose nucleic acid 3.1(pc DNA 3.1)-green fluorescent protein (GFP), so that two novel expression vectors pcDNA 3.1-IE (-94/+52)-GFP and pcDNA 3.1-IE (-945/+52)-GFP which contain prawn cytomegalovirus promoters are constructed. The constructed expression vectors are mixed with a cationic lipofectin transfection reagent TransLipid TM for the intramuscular injection of prawns, results show that the two expression vectors can well express prawn cells, and moreover, host prawns do not have any obvious toxic action. Consequently, the two expression vectors can be used for researching prawn oral vaccines or prawn cytobiology.
Description
Technical field
The present invention relates to the disease-resistant drug development of the biological study of prawn cellular elements and prawn field, specifically two prawn new expression vector construction processs.
Background technology
Environment of Litopenaeus vannamei Low (Litopenaeus vannamei) is occupied very consequence in China's mariculture industry.Yet because the wide-scale distribution of prawn ' s virus, cause the financial loss of tens billion of units to shrimp culture industry every year, has a strong impact on the shrimp culture industry Sustainable development.Although obtained greater advance at prawn etiological diagnosis technical elements at present, aspect prevention and cure of viruses, still do not had good terms of settlement.The prawn antiviral still is in conceptual phase, main direction of studying has subunit vaccine, nucleic acid vaccine, yolk antibody, herbal medicine etc., wherein, the research report is more in subunit vaccine side, VP28 albumen is expressed in the different hosts expression systems such as silkworm pupa, insect cell, Pichia yeast, intestinal bacteria, subtilis, by abdominal muscle injection or oral route, find that expression product has the effect that improves immunizing power and anti-WSSV infection to japonicus, tigar prawn, Procambius clarkii etc.Meng Xiaolin etc. are cloned into expression vector pcDNA3.1 with the WSSV capsid protein gene, obtain recombinant expression vector pcDNA-VP28, this expression vector transforms Salmonellas (Salmonella typhimurium) and enters in the prawn body by oral route, and makes prawn produce disease resistance to WSSV.Attack the poison experiment and show that 25 days protection ratio of the 7th ﹑ 15 ﹑ reaches respectively 83.3% ﹑, 66.7% ﹑ 56.7% behind the oral genetic engineering bacterium, the genetic engineering bacterium oral administration can be survived 7-10 days after entering gut of shrimp, and the host prawn is without any obvious toxic action simultaneously.
Promotor is an integral part of gene (gene), and controlling gene is expressed the time of origin of (transcribing) and the degree of expression.Promotor (Promoters) determines the activity of gene just as " switch ".Promotor not controlling gene itself is movable, but by be called this protein (proteins) of transcribing (transcription) factor in conjunction with and the controlling gene activity.Transcription factor is being commanded the activity of enzyme (enzymes) (RNA polymerase polymerases) just as one side " flag ".This kind of enzyme is being made the rna replicon basis of gene.
Prawn ' s virus WSSV has three early promoters (IE1, IE2, IE3), and wherein the startup ability of IE1 is the strongest, and it all worked in the whole virus infection cycle.There are some researches prove that the IE1 promotor has very strong activity at Insect cells Sf9, the latter is used to the WSSV gene at the expression study of each cell stage, although the Sf9 non-susceptibility cell that is WSSV.IE1 has a Cys2/His2 type zinc fingers, is the characteristic parts of promoter activity.The recombination bacillary viral vector of IE promotor construction expression influenza virus HA gene such as Fang He, host cell is Sf9.Research is found: with respect to the CMV promotor, the ability to express of recombinant baculovirus in cell under the control of IE promotor is enhanced.Wang Yu etc. utilize mETL promotor and the baculovirus polyhedrin body promotor P of m IE1 promotor, baculovirus ETL promotor and the lengthening thereof of IE1 promotor and brachymemma thereof in order to compare the activity of different promoters in baculovirus/insect cell
PHMake up the recombinant baculovirus that contains the EGF P reporter gene under the different promoters control, infected respectively S f9 insect cell, utilized the expression level of Flow cytometry reporter gene.The result shows that the m E T L promotor of the IE1 promotor of WSSV and baculovirus all has promoter activity by force and early in insect cell S f 9 cells, can efficiently express in early days by the control report gene, and P
PHJust show strong active in the infection later stage.
PcDNA3.1-GFP is a carrier for expression of eukaryon, and its promotor is the CMV promotor.This carrier can at multiple Mammals (people, pig, mouse etc.) cell and nonmammalian (fish, insect etc.) cell expressing, therefore be widely used in the RESEARCH ON CELL-BIOLOGY of various animals even the research and development of vaccine.Still there is not at present the report about utilizing the IE1 promotor that pcDNA3.1 is transformed.
Summary of the invention
The purpose of this invention is to provide a kind of two prawn new expression vector construction processs.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
Two prawn new expression vector construction processs, operation steps is as follows:
Two prawn new expression vector construction processs is characterized in that two prawn new expression vectors are double-stranded cyclic DNAs, obtain by transforming carrier for expression of eukaryon PCDNA3.1, and method is as follows:
1. design of primers is with synthetic:
With reference to the synthetic WSSV early genes promotor IE(-94/+52 of WSSV genome sequence among the GenBanK) and IE(-945/+52) primer, see Table 1:
Table 1 primer
Tab.1Primer
Primer is synthetic by China large gene biological Engineering Co., Ltd;
2.IE(-94/+52) and amplification IE(-945/+52):
25.0 μ L systems are adopted in the PCR reaction: WSSV dna profiling 2 μ L, 2xPCR mix 12.5 μ L, each 1 μ L of upstream and downstream primer (25 μ mol/L), ultrapure water 8.5 μ L.IE(-94/+52) PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 35sec, 35 circulations; 72 ℃ of 10min, IE(-945/+52) the PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30sec, 51 ℃ of 30sec, 72 ℃ of 35sec, 35 circulations; 72 ℃ of 10min;
Purpose fragment IE(-94/+52) and clone IE(-945/+52) and conversion 3.:
The PCR product reclaims the test kit explanation by gel to carry out being connected with the PEASY-T1 cloning vector after the purpose fragment reclaims: PCR product 4 μ L, and PEASY-T1 cloning vector 1 μ L, after mixing gently, room temperature is placed 10min, after the reaction end reaction tubes is put on ice.Add and connect product in 50 μ L Trans1-T1 chemoreception attitude cells, flick mixing, ice bath 30min.42 ℃ of heat shock 30sec are put 2min on ice immediately.Reactant adds 500 μ L LB nutrient solutions, and 1h is cultivated in 37 ℃ of concussions.It is dull and stereotyped to get nutrient solution 200 μ L bacterium liquid coating LA, cultivates 12 hours for 37 ℃.
4. evaluation recombinant plasmid PEASY-T1-IE(-94/+52), PEASY-T1-IE(-945/+52):
Single bacterial colony on the above-mentioned LA flat board of picking is seeded to 500 μ L LB nutrient solutions, and 3h is cultivated in 37 ℃ of concussions.Get 1 μ L bacterium liquid and carry out the PCR evaluation, method is seen the explanation of PEASY-T1 cloning vector test kit.
5. sequencing and analysis:
Send China large gene sequencing the positive bacterium liquid of bacterium liquid PCR qualification result.With the DNAstar biosoftware sequencing result is analyzed.
6.pcDNA3.1-IE(-94/+52)-GFP and pcDNA3.1-IE(-945/+52)-structure of GFP expression vector:
With restriction enzyme SacI and Mlu l respectively enzyme cut pcDNA3.1-GFP, PEASY-T1-IE(-94/+52), PEASY-T1-IE(-945/+52), glue reclaims purpose fragment pcDNA3.1-GFP, IE(-94/+52), IE(-945/+52).With IE(-94/+52), IE(-945/+52) be connected with pcDNA3.1 – GFP respectively.Method of attachment: pcDNA3.1 – GFP 2 μ L, IE(-94/+52) (or IE(-945/+52)) 10 μ L, T4DNA Ligase 1 μ L, 5 * T4DNA Ligase Buffer, 4 μ L add water and supply 20 μ L.Room temperature connects 10min, and the conversion of connection product and the authentication method of recon are the same.
New expression vector of the present invention proves and can express at the prawn somatocyte, and method of proof is as follows:
In the Eppendorf pipe, add OPti-MEM I Reduced Serum Medium, pcDNA3.1-IE(-94/+52 by 50 μ L:1 μ g ratios)-GFP, the soft mixing, make the DNA mixed solution.In other Eppendorf pipe, add OPti-MEM I Reduced Serum Medium and TransLipid by 50 μ L:2 μ L ratios
TM, the soft mixing, make TransLipid
TMDiluent, room temperature left standstill 5 minutes.With carrier DNA mixed solution and TransLipid
TMDiluent mixes, and room temperature left standstill 20 minutes, then by the amount intramuscular injection Penaeus vannamei of 50 μ L/ tails, injects altogether prawn 30 tails.PcDNA3.1-IE(-945/+52)-GFP in the same way with OPti-MEM I Reduced Serum Medium, TransLipid
TMMix and the injection prawn.Set up the negative control group without DNA, totally 30 tails, every tail shrimp injection OPti-MEM I Reduced Serum Medium and TransLipid
TMDiluent 50 μ L.After injecting the 4th day, get prawn and be organized in the fluorescence microscopy Microscopic observation.Experimental result: pcDNA3.1-IE(-94/+52)-GFP and pcDNA3.1-IE(-945/+52)-GFP all can express at the prawn somatocyte, the results are shown in Figure 6, Fig. 7, Fig. 8.Each group residue prawn of not getting tissue occurs in rear 30 days dead in injection, get prawn muscle tissue after 30 days, extracts DNA, carries out respectively PCR and detects, and detection method is with step 2.Detected result: injection pcDNA3.1-IE(-94/+52)-GFP group prawn PCR detection IE(-94/+52) be positive, injection pcDNA3.1-IE(-945/+52)-GFP group prawn PCR detects IE(-945/+52) be positive, detect IE(-94/+52 without DNA negative control group group prawn PCR), E(-945/+52) all be negative.Experimental result explanation pcDNA3.1-IE(-94/+52)-GFP and pcDNA3.1-IE(-945/+52)-GFP can retention time be not less than 30 days in the prawn body.
Beneficial effect of the present invention:
The present invention is transformed into pcDNA3.1-IE(-94/+52 with carrier for expression of eukaryon pcDNA3.1 – GFP)-GFP, pcDNA3.1-IE(-945/+52)-carrier preparation method that these two kinds of GFP can express at the prawn somatocyte.Utilize WSSV virus early promoter to transform the PCDNA3.1 expression vector, obtains a kind of new expression vector that can exist at the prawn cell inner stablity, for next step prawn oral vaccine research provides basic.
Description of drawings
Fig. 1 is pcr amplification WSSV promotor IE(-94/+52 of the present invention) electrophoresis result figure.
Fig. 2 is pcr amplification WSSV promotor IE(-945/+52 of the present invention) electrophoresis result figure.
Fig. 3 is PEASY-T1-IE(-94/+52 of the present invention) qualification result figure.
Fig. 4: be PEASY-T1-IE(-945/+52 of the present invention) qualification result figure.
Fig. 5: be pcDNA3.1-IE(-94/+52 of the present invention)-GFP and pcDNA3.1-IE(-945/+52)-structure of GFP expression vector figure as a result.
Fig. 6: pcDNA3.1-IE(-94/+52)-the GFP expression vector is at the somatic expression of results figure of prawn.
Fig. 7: pcDNA3.1-IE(-94/+52)-the GFP expression vector is at the somatic expression of results figure of prawn.
Fig. 8 is without the somatic expression of results figure of DNA negative control group prawn.
Embodiment
Below in conjunction with accompanying drawing and concrete experiment the present invention is further described in detail.
Fig. 1 is pcr amplification WSSV promotor IE(-94/+52 of the present invention) electrophoresis result figure.
Among the figure: Fig. 1 PCR method is: with reference to the synthetic WSSV early genes promotor IE(-94/+52 of WSSV genome sequence among the GenBanK) the upstream and downstream primer, see Table 1.PCR method amplification WSSV promotor IE(-94/+52).Get 10 μ L PCR products and carry out agarose gel electrophoresis.The M swimming lane is dna molecular marker, and 1 swimming lane is IE(-94/+52).The result: IE(-94/+52) stripe size is 146bp, and is consistent with expected results, illustrates that amplification has obtained the IE(-94/+52 of WSSV).
Fig. 2 is pcr amplification WSSV promotor IE(-945/+52 of the present invention) electrophoresis result figure.
Among the figure: Fig. 2 PCR method is: with reference to the synthetic WSSV early genes promotor IE(-945/+52 of WSSV genome sequence among the GenBanK) the upstream and downstream primer, see Table 1.PCR method amplification WSSV promotor IE(-945/+52).Get 10 μ L PCR products and carry out agarose gel electrophoresis.The M swimming lane is dna molecular marker, and 1 swimming lane is IE(-945/+52).The result: IE(-945/+52) stripe size is 997bp, and is consistent with expected results, illustrates that amplification has obtained the IE(-945/+52 of WSSV).
Fig. 3 is PEASY-T1-IE(-94/+52 of the present invention) qualification result figure.
Among the figure: Fig. 3 method: with IE(-94/+52) be connected with cloning vector PEASY-T1, then will connect product transformed competence colibacillus bacterium, be coated with the LA of section plate.Whether observation LA flat board is grown and bacterial colony is arranged (clone's spot) after 12 hours.The some single bacterium colonies of picking carry out bacterium liquid PCR behind 37 ℃ of cultivation 3h and identify to new LB substratum.Get 10 μ L PCR products and carry out agarose gel electrophoresis.The M swimming lane is dna molecular marker, and 1,2,3 swimming lanes are three bacterium colony sample bacterium liquid PCR products.1,2,3 swimming lane amplified fragments sizes and IE(-94/+52 as a result) clip size is consistent.Feeding sample 1 checks order, and analyzes the IE(-94/+52 of sequencing result and NC BI announcement through DNAstar) in full accord.IE(-94/+52 is described) clone successfully.
Fig. 4: be PEASY-T1-IE(-945/+52 of the present invention) qualification result figure.
Among the figure: Fig. 4 method: with IE(-945/+52) be connected with cloning vector PEASY-T1, then will connect product transformed competence colibacillus bacterium, be coated with the LA of section plate.Whether observation LA flat board is grown and bacterial colony is arranged (clone's spot) after 12 hours.The some single bacterium colonies of picking carry out bacterium liquid PCR behind 37 ℃ of cultivation 3h and identify to new LB substratum.Get 10 μ L PCR products and carry out agarose gel electrophoresis.The M swimming lane is dna molecular marker, and 1,2,3 swimming lanes are three bacterium colony sample bacterium liquid PCR products.1,2,3 swimming lane amplified fragments sizes and IE(-945/+52 as a result) clip size is consistent.Feeding sample 1 checks order, and analyzes the IE(-945/+52 of sequencing result and NC BI announcement through DNAstar) in full accord.IE(-945/+52 is described) clone successfully.
Fig. 5: be pcDNA3.1-IE(-94/+52 of the present invention)-GFP and pcDNA3.1-IE(-945/+52)-structure of GFP expression vector figure as a result.
Among the figure: Fig. 5 expression vector establishment method: with IE(-94/+52), IE(-945/+52) carry out respectively SacI and Mlu l double digestion with pcDNA3.1 – GFP, then glue reclaims the purpose fragment.With IE(-94/+52), IE(-945/+52) be connected respectively with pcDNA3.1 – GFP.Connect product through conversion, coated plate, choose spot, PCR and identify after, select the positive colony bacterium to continue enlarged culturing, extracting plasmid, and plasmid carried out SacI and Mlu l double digestion is identified.Qualification result is seen Fig. 5.PcDNA3.1-IE(-94/+52)-obtain the band that two sizes are respectively 6148bp, 146bp behind the GFP double digestion; 6148bp clip size and pcDNA3.1-GFP size match, 146bp fragment and IE(-94/+52) clip size coincide.PcDNA3.1-IE(-945/+52)-obtain the band that two sizes are respectively 6148bp, 997bp behind the GFP double digestion.6148bp clip size and pcDNA3.1 – GFP size match 997bp fragment and IEIE(-945/+52) clip size coincide.PcDNA3.1-IE(-94/+52 is described)-GFP and pcDNA3.1-IE(-945/+52)-the successfully constructing of GFP expression vector.
Fig. 6: pcDNA3.1-IE(-94/+52)-the GFP expression vector is at the somatic expression of results figure of prawn.
Among the figure: in the Eppendorf pipe, add OPti-MEM I Reduced Serum Medium, pcDNA3.1-IE(-94/+52 by 50 μ L:1 μ g ratios)-GFP, the soft mixing, make the DNA mixed solution.In other Eppendorf pipe, add OPti-MEM I Reduced Serum Medium and TransLipid by 50 μ L:2 μ L ratios
TM, the soft mixing, make TransLipid
TMDiluent, room temperature left standstill 5 minutes.With carrier DNA mixed solution and TransLipid
TMDiluent mixes, and room temperature left standstill 20 minutes, then by the amount intramuscular injection Penaeus vannamei of 50 μ L/ tails, injects altogether prawn 30 tails.After injecting the 4th day, get prawn and be organized in the fluorescence microscopy Microscopic observation.The microscopic examination result, the prawn somatocyte sends strong green fluorescence.
Fig. 7: pcDNA3.1-IE(-94/+52)-the GFP expression vector is at the somatic expression of results figure of prawn.
Among the figure: in the Eppendorf pipe, add OPti-MEM I Reduced Serum Medium, pcDNA3.1-IE(-945/+52 by 50 μ L:1 μ g ratios)-GFP, the soft mixing, make the DNA mixed solution.In other Eppendorf pipe, add OPti-MEM I Reduced Serum Medium and TransLipid by 50 μ L:2 μ L ratios
TM, the soft mixing, make TransLipid
TMDiluent, room temperature left standstill 5 minutes.With carrier DNA mixed solution and TransLipid
TMDiluent mixes, and room temperature left standstill 20 minutes, then by the amount intramuscular injection Penaeus vannamei of 50 μ L/ tails, injects altogether prawn 30 tails.After injecting the 4th day, get prawn and be organized in the fluorescence microscopy Microscopic observation.The microscopic examination result, the prawn somatocyte sends than PaleGreen fluorescence.
Fig. 8 is without the somatic expression of results figure of DNA negative control group prawn.
Among the figure: every tail shrimp injection OPti-MEM I Reduced Serum Medium and TransLipid
TMDiluent 50 μ L,, totally 30 tails, inject the 4th day after, get prawn and be organized in the fluorescence microscopy Microscopic observation.The microscopic examination result, prawn somatocyte redgreen fluorescence.
New expression vector pcDNA3.1-IE(-94/+52)-GFP, pcDNA3.1-IE(-945/+52)-mechanism of action of GFP analyzes theoretically and is: the susceptible host of WSSV virus is Penaeus vannamei, this virus up to 100%, is virus the most common in the prawn culturing, that harm is the most serious to the lethality rate of Penaeus vannamei.WSSV can copy at the prawn somatocyte, and early promoter has play a part important.WSSV has three early promoters (IE1, IE2, IE3), and wherein the startup ability of IE1 is the strongest, and it all worked in the whole virus infection cycle.IE1 there are some researches prove that except the prawn cell works again the IE1 promotor also has very strong activity at Insect cells Sf9, and the latter is used to the WSSV gene at the expression study of each cell stage.IE1 has a Cys2/His2 type zinc fingers, is the characteristic parts of promoter activity.The recombination bacillary viral vector of IE promotor construction expression influenza virus HA gene such as Fang He, host cell is Sf9.Research is found: with respect to the CMV promotor, the ability to express of recombinant baculovirus in cell under the control of IE promotor is stronger.Wang Yu etc. utilize mETL promotor and the baculovirus polyhedrin body promotor P of m IE1 promotor, baculovirus ETL promotor and the lengthening thereof of IE1 promotor and brachymemma thereof in order to compare the activity of different promoters in baculovirus/insect cell
PHMake up the recombinant baculovirus that contains the EGF P reporter gene under the different promoters control, infected respectively S f9 insect cell, utilized the expression level of Flow cytometry reporter gene.The result shows that the IE1 promotor of WSSV and the mELT promotor of baculovirus all have promoter activity by force and early in the Insect cells Sf9 cell, can efficiently express in early days by the control report gene, and P
PHJust show strong active in the infection later stage.And find in the research that baculovirus homology iteron (h r1) can strengthen the activity of ELT promotor.
PcDNA3.1-GFP is a carrier for expression of eukaryon, and its promotor is the CMV promotor.This carrier can at multiple Mammals (people, pig, mouse etc.) cell and nonmammalian (fish, insect etc.) cell expressing, therefore be widely used in the RESEARCH ON CELL-BIOLOGY of various animals even the research and development of vaccine.PcDNA3.1 has certain broad applicability to eukaryotic kind, the IE1 promotor is at the Preference that has of prawn cell, infer thus, replace CMV promotor among the pcDNA3.1-GFP with the IE1 promotor, might obtain a kind of expression vector that can efficiently express at the prawn somatocyte.Prove that by a series of related experiment the IE1 promotor that two length is different is replaced two new expression vectors that the CMV promotor of pcDNA3.1-GFP obtains and can be expressed at the prawn somatocyte.
Still there is not at present the report about utilizing the IE1 promotor that pcDNA3.1 is transformed.PcDNA3.1-IE(-94/+52 of the present invention)-GFP, pcDNA3.1-IE(-945/+52)-GFP obtains by following concrete steps:
(1) experiment material: Taq archaeal dna polymerase MIX, T4 ligase enzyme, PEASY-T1 cloning vector, cationic-liposome transfection reagent TransLipid
TM, Trans1-T1 chemoreception attitude cell etc. is available from the Beijing Quanshijin Biotechnology Co., Ltd, restriction enzyme (SacI and Mlu l) is available from precious biotechnology (Dalian) company limited, pcDNA3.1-GFP, WSSV(white spot syndrome virus (WSSV)) to be preserved by this research department, Trypsin, peptone yeast powder, agarose are available from Britain OXOID company.(body weight 10.2 ± 0.33g) is taken from Penaeus vannamei country of Guangxi aquatic products institute seed multiplication farm to the SPF Penaeus vannamei.
(2) IE(-94/+52) amplification: with reference to the synthetic WSSV early genes promotor IE(-94/+52 of WSSV genome sequence among the GenBanK) the upstream and downstream primer sees Table 1.PCR reaction system: WSSV dna profiling 2 μ L, 2 х PCR mix, 12.5 μ L, IE(-94/+52) each 1 μ L of upstream and downstream primer (25 μ mol/L), ultrapure water 8.5 μ L.PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 35sec, 35 circulations; 72 ℃ of 10min.Get 10 μ L PCR products and carry out agarose gel electrophoresis.The M swimming lane is dna molecular marker, and 1 swimming lane is IE(-94/+52).The result: IE(-94/+52) stripe size is 146bp, and is consistent with expected results, illustrates that amplification has obtained the IE(-94/+52 of WSSV).
(3) IE(-945/+52) amplification: with reference to the synthetic WSSV early genes promotor IE(-945/+52 of WSSV genome sequence among the GenBanK) the upstream and downstream primer sees Table 1.PCR reaction system: WSSVDNA template 2 μ L, 2xPCR mix 12.5 μ L, IE(-945/+52) each 1 μ L of upstream and downstream primer (25 μ mol/L), ultrapure water 8.5 μ L.PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30sec, 51 ℃ of 30sec, 72 ℃ of 35sec, 35 circulations; 72 ℃ of 10min.Get 10 μ L PCR products and carry out agarose gel electrophoresis.The M swimming lane is dna molecular marker, and 1 swimming lane is IE(-945/+52).The result: IE(-945/+52) stripe size is 997bp, and is consistent with expected results, illustrates that amplification has obtained the IE(-945/+52 of WSSV).
(4) IE(-94/+52) and clone IE(-945/+52): with IE(-94/+52) mix by the 1:4 volumetric ratio with cloning vector PEASY-T1, effect is 8 minutes under the room temperature, then mixture is added to 50 μ L competent cells, mixing gently, placed 42 ℃ of thermal shocks of water-bath 1 minute, mixture is added to new LB substratum, and 37 ℃ of shaking tables were cultivated 3 hours, got 100 μ L mixtures and were coated with the LA of section plate.Whether 12 as a child observed the LA flat board grows and bacterial colony is arranged (clone's spot).The some single bacterium colonies of picking carry out bacterium liquid PCR behind 37 ℃ of cultivation 3h and identify to new LB substratum.PCR reaction system: bacterium liquid 1 μ L, 2xPCR mix 12.5 μ L, IE(-94/+52) each 1 μ L of upstream and downstream primer (25 μ mol/L), ultrapure water 9.5 μ L.PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30sec, 51 ℃ of 30sec, 72 ℃ of 35sec, 35 circulations; 72 ℃ of 10min.Get 10 μ L PCR products and carry out agarose gel electrophoresis.The M swimming lane is dna molecular marker, and 1,2,3 swimming lanes are three bacterium colony sample bacterium liquid PCR products.1,2,3 swimming lane amplified fragments sizes and IE(-94/+52 as a result) clip size is consistent.Feeding sample 1 checks order, and analyzes the IE(-94/+52 of sequencing result and NCBI announcement through DNAstar) in full accord.IE(-94/+52 is described) clone successfully.Identify the IE clone (945/+52) with same step.
(5) evaluation PEASY-T1-IE(-94/+52): with IE(-94/+52) be connected with cloning vector PEASY-T1, then will connect product transformed competence colibacillus bacterium, be coated with the LA of section plate.Whether 12 as a child observed the LA flat board grows and bacterial colony is arranged (clone's spot).The some single bacterium colonies of picking carry out bacterium liquid PCR behind 37 ℃ of cultivation 3h and identify to new LB substratum.PCR reaction system: bacterium liquid 1 μ L, 2xPCR mix 12.5 μ L, IE(-94/+52) each 1 μ L of upstream and downstream primer (25 μ mol/L), ultrapure water 9.5 μ L.PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30sec, 51 ℃ of 30sec, 72 ℃ of 35sec, 35 circulations; 72 ℃ of 10min.Get 10 μ L PCR products and carry out agarose gel electrophoresis.The M swimming lane is dna molecular marker, and 1,2,3 swimming lanes are three bacterium colony sample bacterium liquid PCR products.1,2,3 swimming lane amplified fragments sizes and IE(-94/+52 as a result) clip size is consistent.Feeding sample 1 checks order, and analyzes the IE(-94/+52 of sequencing result and NC BI announcement through DNAstar) in full accord.IE(-94/+52 is described) clone successfully.(6) evaluation PEASY-T1-IE(-945/+52): with IE(-945/+52) be connected with cloning vector PEASY-T1, then will connect product transformed competence colibacillus bacterium, be coated with the LA of section plate.Whether 12 as a child observed the LA flat board grows and bacterial colony is arranged (clone's spot).The some single bacterium colonies of picking carry out bacterium liquid PCR behind 37 ℃ of cultivation 3h and identify to new LB substratum.PCR reaction system: bacterium liquid 1 μ L, 2 х PCR mix, 12.5 μ L, IE(-945/+52) each 1 μ L of upstream and downstream primer (25 μ mol/L), ultrapure water 9.5 μ L.PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30sec, 51 ℃ of 30sec, 72 ℃ of 35sec, 35 circulations; 72 ℃ of 10min.Get 10 μ L PCR products and carry out agarose gel electrophoresis.The M swimming lane is dna molecular marker, and 1,2,3 swimming lanes are three bacterium colony sample bacterium liquid PCR products.1,2,3 swimming lane amplified fragments sizes and IE(-945/+52 as a result) clip size is consistent.Feeding sample 1 checks order, and analyzes the IE(-945/+52 of sequencing result and NCBI announcement through DNAstar) in full accord.IE(-945/+52 is described) clone successfully.(7) pcDNA3.1-IE(-94/+52)-GFP and pcDNA3.1-IE(-945/+52)-structure of GFP expression vector: with IE(-94/+52), IE(-945/+52) carry out respectively SacI and Mlul double digestion with pcDNA3.1 – GFP, then glue reclaims the purpose fragment.With IE(-94/+52), IE(-945/+52) be connected respectively with pcDNA3.1 – GFP.Connect product through conversion, coated plate, choose spot, PCR and identify after, select the positive colony bacterium to continue enlarged culturing, extracting plasmid, and plasmid carried out SacI and Mlu l double digestion is identified.Qualification result is seen Fig. 5.PcDNA3.1-IE(-94/+52)-obtain the band that two sizes are respectively 6148bp, 146bp behind the GFP double digestion; 6148bp clip size and pcDNA3.1-GFP size match, 146bp fragment and IE(-94/+52) clip size coincide.PcDNA3.1-IE(-945/+52)-obtain the band that two sizes are respectively 6148bp, 997bp behind the GFP double digestion.6148bp clip size and pcDNA3.1 – GFP size match 997bp fragment and IE(-945/+52) clip size coincide.PcDNA3.1-IE(-94/+52 is described)-GFP and pcDNA3.1-IE(-945/+52)-the successfully constructing of GFP expression vector.
Verify by the following method pcDNA3.1-IE(-94/+52)-GFP and pcDNA3.1-IE(-945/+52)-whether GFP can be in the somatic expression of prawn:
(1) in the Eppendorf pipe, add OPti-MEMIReduced Serum Medium, pcDNA3.1-IE(-94/+52 by 50 μ L:1 μ g ratios)-GFP, the soft mixing, make the DNA mixed solution.In other Eppendorf pipe, add OPti-MEM I Reduced Serum Medium and TransLipid by 50 μ L:2 μ L ratios
TM, the soft mixing, make TransLipid
TMDiluent, room temperature left standstill 5 minutes.With carrier DNA mixed solution and TransLipid
TMDiluent mixes, and room temperature left standstill 20 minutes, then by the amount intramuscular injection Penaeus vannamei of 50 μ L/ tails, injects altogether prawn 30 tails.After injecting the 4th day, get prawn and be organized in the fluorescence microscopy Microscopic observation.
(2) in the Eppendorf pipe, add OPti-MEMIReduced Serum Medium, pcDNA3.1-IE(-945/+52 by 50 μ L:1 μ g ratios)-GFP, the soft mixing, make the DNA mixed solution.In other Eppendorf pipe, add OPti-MEM I Reduced Serum Medium and TransLipid by 50 μ L:2 μ L ratios
TM, the soft mixing, make TransLipid
TMDiluent, room temperature left standstill 5 minutes.With carrier DNA mixed solution and TransLipid
TMDiluent mixes, and room temperature left standstill 20 minutes, then by the amount intramuscular injection Penaeus vannamei of 50 μ L/ tails, injects altogether prawn 30 tails.After injecting the 4th day, get prawn and be organized in the fluorescence microscopy Microscopic observation.
(3) control group: every tail shrimp injection OPti-MEM I Reduced Serum Medium and TransLipid
TMDiluent 50 μ L,, totally 30 tails, inject the 4th day after, get prawn and be organized in the fluorescence microscopy Microscopic observation.
Experimental result: pcDNA3.1-IE(-94/+52)-GFP and pcDNA3.1-IE(-945/+52)-GFP all can express at the prawn somatocyte, pcDNA3.1-IE(-94/+52)-GFP in prawn cell expressing fluorescence intensity greater than pcDNA3.1-IE(-945/+52)-GFP, control group be can't see fluorescence.The results are shown in Figure 6, Fig. 7, Fig. 8.
Claims (1)
1. two prawn new expression vector construction processs is characterized in that two prawn new expression vectors are double-stranded cyclic DNAs, obtain by transforming carrier for expression of eukaryon PCDNA3.1, and method is as follows:
1) design of primers is with synthetic:
With reference to the synthetic WSSV early genes promotor IE(-94/+52 of WSSV genome sequence among the GenBanK) and IE(-945/+52) primer, see Table 1:
Table 1 primer
Tab,1Primer
Primer is synthetic by China large gene biological Engineering Co., Ltd;
IE(-94/+52) and amplification IE(-945/+52) 2):
25.0 μ L systems are adopted in the PCR reaction: WSSV dna profiling 2 μ L, 2xPCR mix 12.5 μ L, each 1 μ L of upstream and downstream primer (25 μ mol/L), ultrapure water 8.5 μ L; IE(-94/+52) PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 35sec, 35 circulations; 72 ℃ of 10min; IE(-945/+52) PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30sec, 51 ℃ of 30sec, 72 ℃ of 35sec, 35 circulations; 72 ℃ of 10min;
Purpose fragment IE(-94/+52) and clone IE(-945/+52) and conversion 3):
After the PCR product carries out the recovery of purpose fragment by the explanation of gel recovery test kit, be connected with the PEASY-T1 cloning vector: PCR product 4 μ L, PEASY-T1 cloning vector 1 μ L, after mixing gently, room temperature is placed 10min, reaction is put reaction tubes on ice after finishing, add and connect product in 50 μ L Trans1-T1 chemoreception attitude cells, flick mixing, ice bath 30min, 42 ℃ of heat shock 30sec are put 2min on ice immediately; Reactant adds 500 μ L LB nutrient solutions, and 1h is cultivated in 37 ℃ of concussions; It is dull and stereotyped to get nutrient solution 200 μ L bacterium liquid coating LA, cultivates 12 hours for 37 ℃;
4) evaluation recombinant plasmid PEASY-T1-IE(-94/+52), PEASY-T1-IE(-945/+52):
Single bacterial colony on the above-mentioned LA flat board of picking is seeded to 500 μ LB nutrient solutions, and 3h is cultivated in 37 ℃ of concussions, gets 1 μ L bacterium liquid and carries out the PCR evaluation, and method is seen the explanation of PEASY-T1 cloning vector test kit;
5) sequencing and analysis:
Send China large gene sequencing the positive bacterium liquid of bacterium liquid PCR qualification result, with the DNAstar biosoftware sequencing result is analyzed;
6) pcDNA3.1-IE(-94/+52)-GFP and pcDNA3.1-IE(-945/+52)-structure of GFP expression vector:
With restriction enzyme SacI and Mlu 1 respectively enzyme cut pcDNA3.1-GFP, PEASY-T1-IE(-94/+52), PEASY-T1-IE(-945/+52), glue reclaims purpose fragment pcDNA3.1-GFP, IE(-94/+52), IE(-945/+52).With IE(-94/+52), IE(-945/+52) be connected with pcDNA3.1 – GFP respectively, method of attachment: pcDNA3.1 – GFP 2 μ L, IE(-94/+52) (or IE(-945/+52)) 10 μ L, T4DNA Ligase 1 μ L, 5 * T4DNA Ligase Buffer, 4 μ L, add water and supply 20 μ L, room temperature connects 10min, and the conversion of connection product and the authentication method of recon are the same.
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