CN104726498A - Building method and applications of J avian leukosis virus subgroup LTR based linearized eukaryotic expression vectors - Google Patents
Building method and applications of J avian leukosis virus subgroup LTR based linearized eukaryotic expression vectors Download PDFInfo
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Abstract
The invention relates to a building method and applications of J avian leukosis virus subgroup LTR based linearized eukaryotic expression vectors. According to the invention, by taking sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers and taking a JSNT strain as a template, an ALV-J virus LTR linearized vector is obtained through PCR amplification. The building method and applications of J avian leukosis virus subgroup LTR based linearized eukaryotic expression vectors disclosed by the invention not only can be used for researching the replication, translation and pathogenic mechanisms and the like of ALV-J or other retroviruses, and also can realize the rapid cloning and efficient expression of exogenous genes.
Description
Technical field
The present invention relates to a kind of construction and application of the linearizing carrier for expression of eukaryon based on J subgroup avian leucosis virus LTR.This invention not only can be used for studying the copying of ALV-J or other retrovirus, translate, the mechanism such as to cause a disease; And quick clone, the high expression that can realize foreign gene.
Background technology
According to viral envelope proteins antigenicity, interference neutralization test and host range, avian leukosis virus (ALV) can be divided into 10 subgroups (A-J).Different from other ALV, ALV-J infects and mainly causes hematopoetic cell malignancies hyperplasia, immunosuppression, induction myelocytome, the tumours such as vascular tumor.ALV-J provirus genome structure has complete retroviral gene group structure, comprises 5 ' LTR-gag-pol-env-LTR3 ' basic structure.Wherein, LTR is non-coding long terminal repeat, with virus replication, translate closely related; And be proved to be that to have eukaryotic promoter and enhanser active.5 ' LTR not only has RNA polymerase II promoter function, also has enhanser and transcriptional control original paper.3 ' LTR stops, except functional transcription, also having promotor characteristic except having PolyA.Therefore, effectively utilize sequence component in LTR, mechanism such as not only can studying ALV-J virus replication, translate, cause a disease, and can construction of expression vector, for exogenous gene expression and transport.In the structure of traditional expression vector, the method carrier construction often need design alternative restriction enzyme digestion sites, cut by enzyme, connecting, realizes the expression of foreign gene.But sometimes owing to can not find suitable restriction enzyme site, often cause cloning procedure loaded down with trivial details, inefficiency.Therefore, the linearizing carrier for expression of eukaryon based on ALV-J LTR that exploitation does not rely on restriction enzyme can simplify cloning procedure, realizes gene quick clone and expresses.
Summary of the invention
The technical problem that 1, will solve
The object of the invention is a kind of construction and application method being to provide linearizing carrier for expression of eukaryon based on J subgroup avian leucosis virus LTR, for quick clone and the expression of foreign gene.Principle of the present invention and most crucial gordian technique scientifically design the linearized vector and exogenous genetic fragment that amplify containing ALV-J virus LTR, commercial recombinase ExnaseTM II is utilized to cut ligation without enzyme, directly Quick Casting is cloned in vitro, is transformed into intestinal bacteria subsequently and carries out plasmid amplification and qualification; And positive colony transfecting eukaryotic cells is realized genetic expression and transport.
Described primer is as follows:
A, pcr amplification ALV-J virus LTR linearized vector primer:
Upstream primer: 5 ' ggttccgaacgcaatgtaacgggaggct3 ';
Downstream primer: 5 ' gcttgatccacagggcgaccagaatca3 ';
B, pcr amplification foreign gene primer:
Upstream primer: 5 ' ccctgtggatcaagcATGNNNNNNNNNNNNNNN3 ';
Downstream primer: 5 ' attgcgttcggaaccCTANNNNNNNNNNNNNNN3 ';
The invention discloses pcr amplification ALV-J virus LTR linearized vector primer and pcr amplification foreign gene for the conserved sequence of external Quick Casting cloning primer.The invention also discloses a kind of method expressed based on the foreign gene quick clone of ALV-J virus LTR, it is characterized in that with recombinase ExnaseTM II, linearizing ALV-J virus LTR carrier and exogenous genetic fragment PCR primer are cut ligation without enzyme, and direct recombinant clone is transformed into intestinal bacteria.
2, technical scheme
1) design amplification is containing the ALV-J virus linearized vector of LTR and exogenous genetic fragment primer: amplification is positioned at 28 bases after ALV-J env gene end codon containing the linearized vector upstream primer of ALV-J virus LTR; Amplification is positioned at front 27 bases of ALV-Jgag gene start codon containing the linearized vector downstream primer of ALV-J virus LTR; In the upstream primer of amplification foreign gene except 18 base N of external source gene specific, all conservative with the base of amplification containing the linearized vector downstream primer complementation of ALV-J virus LTR with 15; In the downstream primer of amplification foreign gene except 18 base N of external source gene specific, all conservative with the base of amplification containing the linearized vector upstream primer complementation of ALV-J virus LTR with 15.Concrete primer sequence sees attached list 1, is synthesized by Life Technologies Shanghai Thermo Fischer Scient Inc..
2) based on the construction and application of eukaryotic expression linearized vector of ALV-J virus LTR: with containing ALV-J virus JS-NT provirus full-length genome plasmid JSNT and foreign gene for masterplate, utilize primer in table 1, PCR amplifies containing the ALV-J virus eukaryotic expression linearized vector ALV-LTR of LTR and exogenous genetic fragment (accompanying drawing 1, step 1) respectively; And utilize recombinase ExnaseTMII to carry out Quick Casting clone (accompanying drawing 1, step 2); Positive colony can carry out eukaryotic cell transfection, expression identification (accompanying drawing 1, step 3)
3, beneficial effect
The primer of this invention design and the eukaryotic expression linearized vector based on ALV-J virus LTR of structure, not only copy research retrovirus, translate, equimolecular mechanism of causing a disease is significant; And can realize, to the quick clone high expression of foreign gene, there is potential using value.The quick clone strategy based on recombinase ExnaseTM II is incorporated in the present invention.This cloning process does not rely on restriction enzyme and ligase enzyme, enormously simplify the cloning procedure of foreign gene, improves efficiency.The exogenous gene high-efficient carrier for expression of eukaryon under ALV-J virus LTR regulation and control successfully can be obtained by single-wheel PCR and restructuring.
Accompanying drawing explanation
Fig. 1 is based on the construction and application strategy of the eukaryotic expression linearized vector of ALV-J virus LTR
Step 1: with containing ALV-J virus JS-NT provirus full-length genome plasmid JSNT and foreign gene for masterplate, utilize primer in table 1, PCR amplifies containing the ALV-J virus eukaryotic expression linearized vector ALV-LTR of LTR and exogenous genetic fragment respectively; Step 2: utilize recombinase ExnaseTM II to carry out Quick Casting clone; Step 3: positive colony transfecting eukaryotic cells, expression identification.
Fig. 2 electrophoretic analysis PCR primer
Swimming lane 1, the eukaryotic expression linearized vector based on ALV-J virus LTR amplified; Swimming lane 2, the eGFP fragment amplified.
The expression of Fig. 3 eGFP in the carrier for expression of eukaryon based on ALV-J virus LTR
The DF1 cell of A, transfection ALV-LTR-eGFP; B, the normal DF1 cell of untransfected; The 293T cell of C, transfection ALV-LTR-eGFP; D, the normal 293T cell of untransfected.
Embodiment
Content for a better understanding of the present invention, following embodiment gives the example of the construction and application of the eukaryotic expression linearized vector based on ALV-J virus LTR by reference to the accompanying drawings.
Embodiment
1) design amplification is containing the ALV-J virus linearized vector of LTR and eGFP fragment primer: amplification is positioned at 28 bases after ALV-J env gene end codon containing the linearized vector upstream primer of ALV-J virus LTR; Amplification is positioned at front 27 bases of ALV-Jgag gene start codon containing the linearized vector downstream primer of ALV-J virus LTR; In the upstream primer of amplification eGFP fragment except special 18 bases of eGFP fragment, also conservative with the base of amplification containing the linearized vector downstream primer complementation of ALV-J virus LTR with 15; In the downstream primer of amplification eGFP fragment except special 18 bases of eGFP fragment, also conservative with the base of amplification containing the linearized vector upstream primer complementation of ALV-J virus LTR with 15.Concrete primer sequence sees attached list 1, is synthesized by Life Technologies Shanghai Thermo Fischer Scient Inc..
2) based on eukaryotic expression linearized vector and the amplification of eGFP fragment PCR of ALV-J virus LTR: to contain ALV-J virus JS-NT provirus full-length genome plasmid JSNT and pcDNA3.1-EGFP for masterplate, primer described in table 1 is that primer carries out pcr amplification.As the step 1 in Fig. 1.Pcr amplification reaction system is: 40 μ l water, 5 μ l 10 times damping fluids, 1 μ l 10mM dNTP, 1 μ l10 μm ol upstream primer, 1 μ l 10 μm ol downstream primer, JSNT and pcDNA3.1-EGFP of 1 μ l 10ng/ μ l, 1 μ l commercial Phanta Super-Fidel i ty archaeal dna polymerase.Pcr amplification reaction loop parameter is: 95 DEG C of sex change 3 minutes, and carry out 30 circulations (72 DEG C extend 3 minutes for 95 DEG C of sex change 10 seconds, 57 DEG C of annealing 30 seconds) subsequently, last 72 DEG C extend 10 minutes.After PCR terminates, PCR primer carries out electrophoresis in the sepharose of 1%.As shown in Figure 2, wherein swimming lane M represents the electrophoretic analysis figure of reference substance DNA Marker, and wherein swimming lane 1,2 represents respectively based on the ALV-J virus eukaryotic expression linearized vector ALV-LTR (SEQ ID NO.7) of LTR and the electrophoretic analysis figure of eGFP fragment PCR amplified production.
3) eGFP fragment quick clone enters the carrier for expression of eukaryon based on ALV-J virus LTR: the eukaryotic expression linearized vector ALV-LTR based on ALV-J virus LTR of above purifying and eGFP fragment PCR products are carried out recombinant clone under the effect of commercialization recombinase ExnaseTM II.As the step 2 in Fig. 1.Concrete recombining reaction system is as follows: the eGFP product 50-100ng of purifying, and the eukaryotic expression linearized vector 50ng based on ALV-J virus LTR of purifying, 2 μ l commercial ExnaseTM II enzyme, the damping fluid of 4 μ l 5 times, other benefit adds water to 20 μ l.Reactant after 30 minutes, puts 5 minutes in 37 DEG C of effects on ice.Subsequently 20 μ l reactants are transformed into conventional competence bacterium, are coated with LB plate.Next day, picking bacterial clone carried out plasmid preparation, positive clone identification.
4) expression of eGFP in the carrier for expression of eukaryon based on ALV-J virus LTR: by positive colony (called after ALV-J-LTR-eGFP) the transfection DF1 containing EGFP of acquisition or 293T cell, transfection 12, after 24,36hr, in the expression of fluorescence microscopy Microscopic observation EGFP.As the step 3 in Fig. 1.Concrete transfection procedure is as follows: the ALV-J-LTR-eGFP plasmid getting 2ug joins the OPTI-MEM substratum of 50ul, mixes subsequently with the Mirus transfection reagent of 4ul; After room temperature places 45min, after adding the OPTI-MEM substratum mixing of 450ul, be directly added to adherent DF1 or the 293T cell of fresh culture.After transfection 12hr, in the expression of fluorescence microscopy Microscopic observation EGFP.12 hours after transfection, no matter be at DF1 cell or at 293T cell, the expression of a large amount of eGFP can be observed under fluorescent microscope.Fig. 3 A is the expression of ALV-J-LTR-eGFP in DF1 cell, and Fig. 3 C is the expression of ALV-J-LTR-eGFP in 293T cell; Fig. 3 B, D are normal DF1 and 293T cell respectively.
Table 1 linearized vector and foreign gene design of primers.
Claims (2)
1. based on a preparation method for the linearizing carrier for expression of eukaryon of J subgroup avian leucosis virus LTR, it is characterized in that, utilize following primer, with JSNT strain for template, carry out pcr amplification and obtain ALV-J virus LTR linearized vector;
Upstream primer: 5 ' ggttccgaacgcaatgtaacgggaggct3 ';
Downstream primer: 5 ' gcttgatccacagggcgaccagaatca3 '.
2. utilize a method for the linearizing carrier for expression of eukaryon expression alien gene of J subgroup avian leucosis virus LTR, it is characterized in that comprising the following steps:
1) to contain ALV-J virus provirus full-length genome plasmid JSNT for template, with SEQ IDNO.1 and 2 for primer, pcr amplification goes out the eukaryotic expression linearized vector containing ALV-J virus LTR;
2) take foreign gene as template, with SEQ IDNO.3 and 4 for primer, pcr amplification goes out exogenous genetic fragment;
3) and utilize recombinase ExnaseTM II to step 1) and 2) PCR primer that obtains carries out Quick Casting clone; Positive colony can carry out eukaryotic cell transfection, expression identification.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190093171A (en) * | 2018-01-31 | 2019-08-08 | 서울대학교산학협력단 | A method for producing avian leukosis virus(alv)-resistant avian line using crispr/cas9 system |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660581A (en) * | 2003-01-24 | 2012-09-12 | 辛那杰瓦生物制药股份有限公司 | Production of transgenic avian species |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102660581A (en) * | 2003-01-24 | 2012-09-12 | 辛那杰瓦生物制药股份有限公司 | Production of transgenic avian species |
Non-Patent Citations (4)
| Title |
|---|
| 叶建强: ""J亚群禽白血病病毒Env蛋白分子生物学特性研究"", 《中国博士学位论文全文数据库农业科技辑》 * |
| 张伟伟等: ""J亚群禽白血病病毒的LTR体外启动活性分析"", 《中国兽医科学》 * |
| 深海的鱼1973: ""ClonExpress一步法定向克隆无缝克隆试剂盒说明书"", 《百度文库》 * |
| 王蓓等: ""以J 亚型禽白血病病毒LTR 为启动子的真核表达载体构建"", 《中国预防兽医学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190093171A (en) * | 2018-01-31 | 2019-08-08 | 서울대학교산학협력단 | A method for producing avian leukosis virus(alv)-resistant avian line using crispr/cas9 system |
| KR102136132B1 (en) | 2018-01-31 | 2020-07-22 | 서울대학교 산학협력단 | A method for producing avian leukosis virus(alv)-resistant avian line using crispr/cas9 system |
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