CN101343638A - Toxoplasma transgenosis carrier based on piggyback transposon and construction method - Google Patents
Toxoplasma transgenosis carrier based on piggyback transposon and construction method Download PDFInfo
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Abstract
The invention provides a toxoplasma gene transfer vector which is based on piggyBac transposon and a preparation method thereof. The toxoplasma gene transfer vector is composed of a piggyBac transposon expression vector and an auxiliary plasmid for expressing piggyback transposase. The piggyBac transposon expression vector is constructed of toxoplasma promoter sequences such as the regulating elements of tachyzoite and bradyzoite specific expressing protein SAG1, BAG1 and GRA, purpose genes as well as the piggyBac inverted repetitive sequence; the auxiliary plasmid is constructed of the regulating elements of the SAG1 and the GRA and piggyBac transposase coded genes. Compared with the prior art, the invention has the advantages that the operation is simple, and the conversion efficiency is high, thereby providing a new path for the research and certification on the insect drug target and vaccine candidate antihelion against toxoplasma.
Description
Technical field
The present invention aims to provide a kind of toxoplasma cdna transfer vector based on the piggyBac transposon, also discloses this construction of carrier simultaneously, belongs to biological technical field.
Background technology
Toxoplasmosis (Toxoplasmosis) is to suffer from parasitosis altogether by a kind of universal people beast that toxoplasma gondii (Toxoplasma gondii) causes.Human arch insect infection rate is high, can cause serious disease in some cases, can influence the growth of fetus as pregnant woman's toxoplasma gondii infection, and severe patient teratogenesis even death can make the pregnant woman miscarry or premature labor simultaneously; For immunosuppression or immune deficiency patient, toxoplasma gondii is a main opportunistic paathogenic factor.There is 30%～46% probability to propagate to fetus behind pregnant woman's toxoplasma gondii infection, causes that fetal congenital infects, and causes miscarriage, stillborn foetus or congenital toxoplasmosis from mother; 6%～10% the concurrent toxoplasmosis of AIDS patient is arranged, and acquired immune deficiency syndrome (AIDS) encephalitis that patient suffers from has 50% to be caused by arch insect infection.According to investigations, pregnant and lying-in women and tumour patient toxoplasma antibody positive rate are up to 10%～60%.Animal infection toxoplasma gondii such as cat, dog, pig, sheep, ox, rabbit are very general, and its infection rate not only causes the domestic animal miscarriage up to 10%～50%, influences livestock industry production, and becomes the main contagium of human toxoplasmosis.Therefore, toxoplasmosis has become one of important public hygiene problem that China needs to be resolved hurrily.Toxoplasmosis does not still have ideal control medicine so far, owing to can cause host's protective immune response behind the arch insect infection, therefore developing safe and effective vaccine should be toxoplasmosis better prevention countermeasure.
Toxoplasma cdna group order-checking is in recent years finished, and part toxoplasma gondii virulence associated gene and some antigen genes have been identified, for the drug development and the vaccine research of toxoplasma gondii provides some candidate's targets, but still have a large amount of functional genes to need to identify, grow regulatory gene, other important virulence gene etc. as toxoplasma gondii.
Identify the functional gene of toxoplasma gondii, at first need to make up its mutant, present operable method comprises that chemomorphosis, gene targeting, RNA disturb technology such as (RNAi), and these methods respectively have relative merits.Utilization chemomorphosis method makes up toxoplasma gondii Tbd-mutant, shows as bradyzoite formation ability and weakens, but can not determine its molecule mechanism; Identified part virulence, the metabolism related gene of toxoplasma gondii by gene targeting, but this method once can only make up a mutant, and efficient is not high, workload is very big.The RNAi technology is also very complicated in operation, at first needs to design a large amount of siRNA molecules according to target-gene sequence, verifies its gene silencing effect then one by one, and also be difficult to reach gene silencing completely on effect.
The piggyBac transposon is the DNA transposon that derives from lepidopterous insects cabbage looper (Trichoplusia ni), total length 2.476kbp, contain 1 rna plymerase ii promoter region and 1 polyadenylic acid signal, the side of this signal is 1 about 2.1kb opening code-reading frame, the transposase of 1 68kDa of coding.The end of piggyBac transposon is the inverted repeats of long 13bp, the distributing inside inverted repeats of 19bp of two ends asymmetricly.5 ' end of piggyBac transposon inverted repeats contains 2-3 C base, and 3 ' terminal G base works in cutting out the chosen process in site.This transposon swivel base frequency height, and always insert in the TTAA site, be included into the transferable factor family of TTAA.As a kind of gene mutagenesis instrument, the piggyBac transposon has higher swivel base efficient, can produce a large amount of stable mutant that have single piggyBac transposon that go down to posterity fast and efficiently, thereby avoid traditional mutafacient system such as gene targeting, save cost greatly; Loading capacity is big, can carry the large fragment DNA that contains a plurality of genes simultaneously; Transgenosis can be expressed steadily in the long term; And integrate the expression situation of easier simulation native gene with single copy form; Utilize inverse PCR can determine easily that piggyBac inserts genomic position, thereby determine gene function; PiggyBac can extensively insert in the genome, and especially the preference gene coding region effectively destroys the native gene function, produces similar phenotype with traditional gene targeting mutagenesis; That utilizes piggyBac accurately cuts off the recovery that can realize mutant phenotype, thereby further determines the corresponding relation of phenotype and gene.What is more important, the species specificity of piggyBac transposon system is poor, except insect, all shows higher transposition activity mammalian cell, Schistosoma mansoni and plasmodium, and this provides foundation for its widespread use.
Summary of the invention
The present invention aims to provide a kind of toxoplasma cdna transfer vector based on the piggyBac transposon, and the research that is used for toxoplasma gondii drug targets and vaccine candidate antigen is identified.
The invention also discloses the preparation method of above-mentioned carrier, have the characteristics simple, that transformation efficiency is high that make up.
Toxoplasma cdna transfer vector based on the piggyBac transposon provided by the invention is characterized in that: the helper plasmid that comprises piggyBac transposon expression vector and expression piggyBac transposase.
Described toxoplasma cdna transfer vector is characterized in that: the piggyBac transposon expression vector of toxoplasma cdna transfer vector is the inverted repeats of piggyBac transposon, the specific expressed proteic controlling element of toxoplasma gondii.
Described toxoplasma cdna transfer vector is characterized in that: helper plasmid is that the specific expressed protein regulation element of toxoplasma gondii instructs expression piggyBac transposase down.
Described toxoplasma cdna transfer vector is characterized in that: the inverted repeats of piggyBac transposon and transposase are selected from the insect powder Autographa spp.
Described toxoplasma cdna transfer vector, the specific expressed protein regulation element of toxoplasma gondii that it is characterized in that making up the toxoplasma cdna transfer vector be 5 of 5 of the specific expressed protein SAG of toxoplasma tachyzoite ' end non-coding region and 3 ' end non-coding region, the specific expressed protein SAG of bradyzoite ' end non-coding region and 3 ' end non-coding region, toxoplasma gondii dense granule proteic 5 ' end non-coding region and 3 ' end non-coding region and toxoplasma gondii other is proteic 5 ' hold non-coding region and 3 ' end non-coding region.
The preparation method of the toxoplasma cdna transfer vector based on the piggyBac transposon of the present invention specifically may further comprise the steps:
1) structure of piggyBac transposon expression vector: the promoter sequence that the utilization round pcr is cloned the specific expressed protein SAG 1 of toxoplasma gondii polyadenylic acid signal sequence and tachyzoite or bradyzoite or BAG1 or GRA respectively; Make up toxoplasma gondii destination gene expression box; Be cloned between the piggyBac inverted repeats by the destination gene expression box of gene clone technology, make up toxoplasma gondii piggyBac transposon expression vector above-mentioned structure;
2) structure of helper plasmid: piggyBac transposase encoding gene is cloned into toxoplasma gondii SAG1 or GRA promoter sequence downstream, and structure can be expressed the helper plasmid of transposase.
In order to realize that with stable being incorporated in the toxoplasma cdna group of destination gene expression box the present invention adopts the piggyBac transposon to make up the toxoplasma cdna conversion system.Studies show that the piggyBac transposon can in many lepidopterans, dipteral insect and many mammalian cells swivel base take place, its swivel base mechanism is: two ends of piggyBac transposon respectively have a tumor-necrosis factor glycoproteins, the direction of two tumor-necrosis factor glycoproteinss is opposite, be called reverse terminal repeat, comprise that therebetween two are read frame, swivel base when taking place in one of them transposase of encoding, transposase promotes the swivel base of the dna fragmentation between the two reverse terminal repeats by the reverse terminal repeat of identification.The present invention utilizes the DNA recombinant technology, complete goal gene encoder block expression cassette is cloned between the reverse terminal repeat of piggyBac transposon, thereby transform the piggyBac transposon as gene transfer vector, gene transfer vector can stably be incorporated into reporter gene in the toxoplasma cdna group.
Transposon generation swivel base also needs the participation of transposase except that needing reverse terminal repeat.Transposase is discerned reverse terminal repeat, and realize two reverse terminal repeats and inner dna fragmentation generation swivel base thereof, for the stable gene of realizing swivel base is incorporated in the toxoplasma cdna group, the present invention is cloned into transposase gene in another carrier, and add toxoplasma tachyzoite specificity SAG1 gene promoter at its upstream, thereby be built into can transient expression the helper plasmid of transposase gene, guaranteed that the swivel base successful reaction carries out.
This two plasmid transgenosis system by the transformation of piggyBac transposon will need the goal gene of swivel base to separate with transposase, not only can realize the high frequency integration of goal gene in the toxoplasma cdna group, and guarantee the genetic stability of integrator gene in the transgenosis toxoplasma gondii.
Positively effect of the present invention is: acquisition by expression vector plasmid and the two toxoplasma cdna transfer system that constitutes jointly of helper plasmid, guarantee the high effective integration of external source goal gene in the toxoplasma cdna group.The present invention compared with prior art, have easy and simple to handle, transformation efficiency is high, identifying for the research of toxoplasma gondii drug targets and vaccine candidate antigen provides new way.
Description of drawings
The walk around construction strategy of stand expression vector of Fig. 1 toxoplasma gondii piggyBac
The construction strategy of Fig. 2 toxoplasma gondii piggyBac transposon system supplymentary matter
The construction strategy of Fig. 3 toxoplasma gondii piggyBac transposon bradyzoite expression vector
The construction strategy of Fig. 4 toxoplasma gondii piggyBac transposon tachyzoite-bradyzoite expression vector
Fig. 5 toxoplasma gondii piggyBac transposon electroporation transfection result
A. toxoplasma gondii piggyBac transposon expression vector and helper plasmid (expression transposase) cotransfection toxoplasma gondii trophont fluorescent microscope detects; B. toxoplasma gondii piggyBac transposon expression vector (PB-) toxoplasma gondii trophont fluorescent microscope detects; C. toxoplasma gondii piggyBac transposon electroporation transfection efficient [PB+:piggyBac transposon expression vector and helper plasmid (expression transposase) cotransfection; PB-: toxoplasma gondii piggyBac transposon expression vector transfection].
Embodiment
Be intended to further illustrate the present invention below by embodiment and concrete operation steps, rather than limit the present invention by any way.Under the prerequisite that does not deviate from the spirit and principles in the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope that awaits the reply of the present invention.
Embodiment 1
The structure of toxoplasma tachyzoite gene transfer vector and application
Prepare the toxoplasma tachyzoite transfer vector according to the technological line shown in the accompanying drawing 1,2, concrete steps are as follows:
1. the clone and the sequential analysis of toxoplasma gondii SAG1 promoter sequence and polyadenylic acid signal sequence
With RH strain of Toxoplasma gondii abdominal cavity inoculation kunming mice, extract ascites after 3-4 days, centrifugal collection polypide, and extract the toxoplasma cdna group according to a conventional method, with SAG1-F and SAG1-R is primer, 5 ' end non-coding region SAG1-5 of utilization PCR method amplification toxoplasma gondii SAG1 gene; With GRA2-F and GRA2-R is primer, 3 ' end non-coding region GRA2-3 of utilization PCR method amplification toxoplasma gondii GRA2 gene, amplified production is after agarose gel electrophoresis is determined, with the gene clone that obtains to the pMD-18T carrier, construction recombination plasmid pMD/SAG1-5 and pMD/GRA2-3 adopt the terminal cessation method of two deoxidations to survey its sequence.
SAG1-F：5′-GGAATTCGCGTGTTCTAACCACAAACCTTG-3′；
SAG1-R：5′-GGTCTAGAGTGGTTCTCCGTCGGTGTGAG-3′；
GRA2-F：5′-GTCTAGAAAACTCGAGAATAATGTTGACTACGACGAAAGTGA-3′；
GRA2-R：5′-GGAAGCTTGTCGACTGGAACTACGGTGTTTG-3′。
2. the structure of toxoplasma gondii expression cassette SAG1/RFP
The amplification of reporter gene RFP is a primer with RFP-F and RFP-R,
RFP-F：5′-GGCTCTAGAATGGCCTCCTCCGAGGACG-3′；
RFP-R：5′-GCTCGAGTTAGGCGCCGGTGGAGTGG-3′
With plasmid PB[Act-RFP] DS is template, amplified fragments is cloned into the T carrier, obtains the complete coding region RFP of RFP gene with the XbaI/XhoI double digestion; Plasmid pMD/SAG1-5 and pMD/GRA2-3 difference EcoRI/XbaI and XhoI/HindIII double digestion release purpose fragment SAG1-5 and GRA2-3 with above-mentioned structure.According to a conventional method these three fragments are connected, obtain toxoplasma gondii expression cassette SAG1/RFP.
3. the structure of toxoplasma gondii piggyBac transposon system expression vector
With the toxoplasma gondii expression cassette SAG1/RFP of above-mentioned structure utilization EcoRI/XhoI/ double digestion, reclaim expression cassette and it be cloned into plasmid PB[Act-RFP] the HindIII/BamHI site of DS, can obtain toxoplasma gondii piggyBac transposon system expression vector.
4. the structure of toxoplasma gondii piggyBac transposon system helper plasmid
As shown in Figure 2, transposase coding region among plasmid CMV-PBase utilization HindIII/EcoRI double digestion is discharged, reclaim the Pbase coding region, and it is cloned into XbaI/XhoI site among the toxoplasma gondii expression cassette SAG1/RFP, promptly obtain to express the helper plasmid SAG1-PBase of transposase.
5. the transfection of toxoplasma gondii
By toxoplasma gondii piggyBac transposon expression vector and the helper plasmid cotransfection RH strain of Toxoplasma gondii trophont of electroporation method with structure;
6. the evaluation of swivel base characteristic
Utilization fluorescent microscope and inverse PCR technical evaluation piggyBac transposon are in the intravital swivel base characteristic of toxoplasma gondii (swivel base efficient, insertion site feature) (Fig. 5);
7. by the limiting dilution method above-mentioned toxoplasma gondii mutant is cultivated through 96 well culture plates, set up the toxoplasma gondii mutant clone, and carry out the evaluation of biology and immunological characteristic.
Embodiment 2
The structure and the application of toxoplasma gondii bradyzoite gene transfer vector
According to the path of preparing toxoplasma gondii bradyzoite transfer vector shown in the accompanying drawing 3, concrete steps are as follows:
1. the clone of toxoplasma gondii BAG1 promoter sequence
With RH strain of Toxoplasma gondii abdominal cavity inoculation kunming mice, extract ascites after 3-4 days, centrifugal collection polypide, and extract the toxoplasma cdna group according to a conventional method, with SAG1-F and SAG1-R is primer, 5 ' end non-coding region SAG1-5 of utilization PCR method amplification toxoplasma gondii SAG1 gene; With GRA2-F and GRA2-R is primer, 3 ' end non-coding region GRA2-3 of utilization PCR method amplification toxoplasma gondii GRA2 gene, amplified production is after agarose gel electrophoresis is determined, with the gene clone that obtains to the pMD-18T carrier, construction recombination plasmid pMD/BAG1-5 and pMD/GRA2-3 adopt the terminal cessation method of two deoxidations to survey its sequence.
BAG1-F：5′-GGATCCTGATCAGTGCACTCTACGAGATGC-3′；
BAG1-R：5′-GGGGGTCTAGACTTTTTTGAATATCATACGGG-3′；
GRA2-F：5′-GTCTAGAAAACTCGAGAATAATGTTGACTACGACGAAAGTGA-3′；
GRA2-R：5′-GGAAGCTTGTCGACTGGAACTACGGTGTTTG-3′。
2. the structure of toxoplasma gondii expression cassette BAG1/RFP
The amplification of reporter gene RFP is a primer with RFP-F and RFP-R
RFP-F：5′-GGCTCTAGAATGGCCTCCTCCGAGGACG-3′；
RFP-R：5′-GCTCGAGTTAGGCGCCGGTGGAGTGG-3′。
With plasmid PB[Act-RFP] DS is template, amplified fragments is cloned into the T carrier, obtains the complete coding region RFP of RFP gene with the XbaI/XhoI double digestion; Plasmid pMD/BAG1-5 and pMD/GRA2-3 difference EcoRI/XbaI and XhoI/HindIII double digestion release purpose fragment BAG1-5 and GRA2-3 with above-mentioned structure.According to a conventional method these three fragments are connected, obtain toxoplasma gondii expression cassette BAG1/RFP.
3. the structure of toxoplasma gondii piggyBac transposon system expression vector
With the toxoplasma gondii expression cassette BAG1/RFP of above-mentioned structure utilization EcoRI/XhoI/ double digestion, reclaim expression cassette and it be cloned into plasmid PB[Act-RFP] the HindIII/BamHI site of DS, can obtain toxoplasma gondii piggyBac transposon system expression vector.
4. the structure of toxoplasma gondii piggyBac transposon system helper plasmid
As shown in Figure 2, transposase coding region among plasmid CMV-PBase utilization HindIII/EcoRI double digestion is discharged, reclaim the PBase coding region, and it is cloned into XbaI/XhoI site among the toxoplasma gondii expression cassette SAG1/RFP, promptly obtain to express the helper plasmid SAG1-PBase of transposase.
5. the transfection of toxoplasma gondii
By toxoplasma gondii piggyBac transposon expression vector and the helper plasmid cotransfection RH strain of Toxoplasma gondii trophont of electroporation method with above-mentioned structure.
6. the evaluation of swivel base characteristic
Utilization fluorescent microscope and inverse PCR technical evaluation piggyBac transposon are in the intravital swivel base characteristic of toxoplasma gondii (swivel base efficient, insertion site feature).
7. by the limiting dilution method above-mentioned toxoplasma gondii mutant is cultivated through 96 well culture plates, set up the toxoplasma gondii mutant clone, and carry out the evaluation of biology and immunological characteristic.
Embodiment 3
The structure and the application of toxoplasma tachyzoite-bradyzoite gene transfer vector
According to the expression vector of the path of preparing toxoplasma tachyzoite-bradyzoite transfer vector shown in the accompanying drawing 4, concrete steps are as follows:
1. the clone of toxoplasma gondii GRA2 gene promoter sequence
With RH strain of Toxoplasma gondii abdominal cavity inoculation kunming mice, extract ascites after 3-4 days, centrifugal collection polypide, and extract the toxoplasma cdna group according to a conventional method, with GRA2P-F and GRA2P-R is primer, 5 ' end non-coding region GRA2-5 of utilization PCR method amplification toxoplasma gondii GRA2 gene; With GRA2-F and GRA2-R is primer, 3 ' end non-coding region GRA2-3 of utilization PCR method amplification toxoplasma gondii GRA2 gene, amplified production is after agarose gel electrophoresis is determined, with the gene clone that obtains to the pMD-18T carrier, construction recombination plasmid pMD/GRA2-5 and pMD/GRA2-3 adopt the terminal cessation method of two deoxidations to survey its sequence.
GRA2P-F：5′-GGAATTCAAACAAGTTCGTCGCAAAAGG-3′
GRA2P-R：5′-CGTCTAGATGTGAGGCGATATGTGGAGAAG-3′
GRA2-F：5′-GTCTAGAAAACTCGAGAATAATGTTGACTACGACGAAAGTGA-3′；
GRA2-R：5′-GGAAGCTTGTCGACTGGAACTACGGTGTTTG-3′。
2. the structure of toxoplasma gondii expression cassette GRA2/RFP
The amplification of reporter gene RFP is a primer with RFP-F and RFP-R
RFP-F：5′-GGCTCTAGAATGGCCTCCTCCGAGGACG-3′；
RFP-R：5′-GCTCGAGTTAGGCGCCGGTGGAGTGG-3′。
With plasmid PB[Act-RFP] DS is template, amplified fragments is cloned into the T carrier, obtains the complete coding region RFP of RFP gene with the XbaI/XhoI double digestion; Plasmid pMD/GRA2-5 and pMD/GRA2-3 difference EcoRI/XbaI and XhoI/HindIII double digestion release purpose fragment GRA2-5 and GRA2-3 with above-mentioned structure.According to a conventional method these three fragments are connected, obtain toxoplasma gondii expression cassette GRA2/RFP.
3. the structure of toxoplasma gondii piggyBac transposon system expression vector
With the toxoplasma gondii expression cassette GRA2/RFP of above-mentioned structure utilization EcoRI/XhoI/ double digestion, reclaim expression cassette and it be cloned into plasmid PB[Act-RFP] the HindIII/BamHI site of DS, can obtain toxoplasma gondii piggyBac transposon system expression vector.
4. the structure of toxoplasma gondii piggyBac transposon system helper plasmid
As shown in Figure 2, transposase coding region among plasmid CMV-PBase utilization HindIII/EcoRI double digestion is discharged, reclaim the PBase coding region, and it is cloned into XbaI/XhoI site among the toxoplasma gondii expression cassette SAG1/RFP, promptly obtain to express the helper plasmid SAG1-PBase of transposase.
5. the transfection of toxoplasma gondii
By toxoplasma gondii piggyBac transposon expression vector and the helper plasmid cotransfection RH strain of Toxoplasma gondii trophont of electroporation method with above-mentioned structure.
6. the evaluation of swivel base characteristic
Utilization fluorescent microscope and inverse PCR technical evaluation piggyBac transposon are in the intravital swivel base characteristic of toxoplasma gondii (swivel base efficient, insertion site feature).
7. by the limiting dilution method above-mentioned toxoplasma gondii mutant is cultivated through 96 well culture plates, set up the toxoplasma gondii mutant clone, and carry out the evaluation of biology and immunological characteristic.
Claims (6)
1. the toxoplasma cdna transfer vector based on the piggyBac transposon is characterized in that: the helper plasmid that comprises piggyBac transposon expression vector and expression piggyBac transposase.
2. toxoplasma cdna transfer vector according to claim 1 is characterized in that: the piggyBac transposon expression vector of toxoplasma cdna transfer vector is the inverted repeats of piggyBac transposon, the specific expressed proteic controlling element of toxoplasma gondii.
3. toxoplasma cdna transfer vector according to claim 1 is characterized in that: helper plasmid is that the specific expressed protein regulation element of toxoplasma gondii instructs expression piggyBac transposase down.
4. according to claim 1,2,3 described toxoplasma cdna transfer vectors, it is characterized in that: the inverted repeats of piggyBac transposon and transposase are selected from the insect powder Autographa spp.
5. according to claim 2,3 described toxoplasma cdna transfer vectors, the specific expressed protein regulation element of toxoplasma gondii that it is characterized in that making up the toxoplasma cdna transfer vector be 5 of 5 of the specific expressed protein SAG of toxoplasma tachyzoite ' end non-coding region and 3 ' end non-coding region, the specific expressed protein SAG of bradyzoite ' end non-coding region and 3 ' end non-coding region, toxoplasma gondii dense granule proteic 5 ' end non-coding region and 3 ' end non-coding region and toxoplasma gondii other is proteic 5 ' hold non-coding region and 3 ' end non-coding region.
6, the preparation method of the described carrier of claim 1 may further comprise the steps:
1) structure of piggyBac transposon expression vector: the promoter sequence that the utilization round pcr is cloned the specific expressed protein SAG 1 of toxoplasma gondii polyadenylic acid signal sequence and tachyzoite or bradyzoite or BAG1 or GRA respectively; Make up toxoplasma gondii destination gene expression box; Be cloned between the piggyBac inverted repeats by the destination gene expression box of gene clone technology, make up toxoplasma gondii piggyBac transposon expression vector above-mentioned structure;
2) structure of helper plasmid: piggyBac transposase encoding gene is cloned into toxoplasma gondii SAG1 or GRA promoter sequence downstream, and structure can be expressed the helper plasmid of transposase.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101659967B (en) * | 2009-09-10 | 2011-09-28 | 浙江大学 | PiggyBac transposon vector for producing transgenic pig and construction method thereof |
| CN102943092A (en) * | 2012-11-20 | 2013-02-27 | 西北农林科技大学 | General type PiggyBac transposon transgenosis carrier and preparation method thereof |
| CN103320501A (en) * | 2012-03-19 | 2013-09-25 | 中国农业科学院上海兽医研究所 | Primer, probe and kit for detecting bradyzoite of toxoplasma gondii |
| CN115176016A (en) * | 2020-02-19 | 2022-10-11 | 上海药明生物技术有限公司 | Enhanced expression systems and methods of use thereof |
-
2008
- 2008-08-20 CN CN200810051107A patent/CN101343638B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101659967B (en) * | 2009-09-10 | 2011-09-28 | 浙江大学 | PiggyBac transposon vector for producing transgenic pig and construction method thereof |
| CN103320501A (en) * | 2012-03-19 | 2013-09-25 | 中国农业科学院上海兽医研究所 | Primer, probe and kit for detecting bradyzoite of toxoplasma gondii |
| CN103320501B (en) * | 2012-03-19 | 2014-09-03 | 中国农业科学院上海兽医研究所 | Primer, probe and kit for detecting bradyzoite of toxoplasma gondii |
| CN102943092A (en) * | 2012-11-20 | 2013-02-27 | 西北农林科技大学 | General type PiggyBac transposon transgenosis carrier and preparation method thereof |
| CN102943092B (en) * | 2012-11-20 | 2014-12-10 | 西北农林科技大学 | General type PiggyBac transposon transgenosis carrier and preparation method thereof |
| CN115176016A (en) * | 2020-02-19 | 2022-10-11 | 上海药明生物技术有限公司 | Enhanced expression systems and methods of use thereof |
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