CN105483156A - Application of Bac-to-Bac baculovirus expression system to preparation of bombyx mori micro RNA overexpression system - Google Patents

Application of Bac-to-Bac baculovirus expression system to preparation of bombyx mori micro RNA overexpression system Download PDF

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CN105483156A
CN105483156A CN201610096040.XA CN201610096040A CN105483156A CN 105483156 A CN105483156 A CN 105483156A CN 201610096040 A CN201610096040 A CN 201610096040A CN 105483156 A CN105483156 A CN 105483156A
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silkworm
bac
pfastbac1
microrna
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刘仕平
何婷
尹权�
吴小燕
黄亚玺
王伟
夏庆友
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Southwest University
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Abstract

The invention discloses application of a Bac-to-Bac baculovirus expression system to preparation of a bombyx mori micro RNA overexpression system. The bombyx mori micro RNA overexpression system contains a recombination pFastBac1vector, and the recombination pFastBac1vector is obtained in the mode that red fluorescent protein coding genes and a secondary precursor sequence are sequentially inserted into pFastBac1vector polyclone enzyme cutting sites. According to the application, the Bac-to-Bac baculovirus expression system is utilized to serve as the bombyx mori micro RNA overexpression system, the overexpression silkworm cluster microRNAs can be achieved on the aspects of silkworm cells and the individual level, and a new tool is provided for functional research of silkworm miRNA.

Description

The application of Bac-to-Bac baculovirus expression system in preparation silkworm microRNA overexpression system
Technical field
The invention belongs to biological technical field, be specifically related to the application of Bac-to-Bac baculovirus expression system in preparation silkworm microRNA overexpression system.
Background technology
Silkworm (silkworm, Bombyxmori) is a kind of lepidopterous insects with Important Economic value and scientific research value.Along with the parsing of domestic silkworm gene group, to the research entered function genome era of silkworm.MiRNA technology is one of technology of conventional functional gene research, the method that current miRNA commonly uses is the miRNA stand-in of hormone synthetic or carrys out high expression silkworm miRNA in body by transgene carrier, but the miRNA stand-in of hormone synthetic, cost is high, phase effect is short; But and carry out by transgene carrier the limitation that high expression silkworm miRNA is subject to expression vector, its reason is that current most of commercialization miRNA expression vector is applicable to mammalian cell, is but not suitable for the expression of miRNA in insect cell.
Therefore, be badly in need of a kind of miRNA expression system being applicable to silkworm, can silkworm efficiently expressing exogenous gene in individual or cell, the functional study for silkworm miRNA provides new instrument.
Summary of the invention
In view of this, an object of the present invention is to provide the application of Bac-to-Bac baculovirus expression system in preparation silkworm microRNA overexpression system.
For achieving the above object, the invention provides following technical scheme:
The application of Bac-to-Bac baculovirus expression system in preparation silkworm microRNA overexpression system.
In the present invention, described silkworm microRNA overexpression system contains restructuring pFastBac1 carrier, and described restructuring pFastBac1 carrier is cut site by pFastBac1 carrier polyclone enzyme and inserted red fluorescent protein encoding gene successively and express the secondary precursor sequence of silkworm microRNA and obtain.
Preferably, described restructuring pFastBac1 carrier inserts red fluorescent protein encoding gene by EcoRI and SpeI of pFastBac1 carrier, and inserts the secondary precursor sequence of expression silkworm microRNA in XbaI and KpnI restriction enzyme site and obtain.
Preferred, the nucleotide sequence of described red fluorescent protein encoding gene is as shown in SEQIDNO.5.
Preferred, the nucleotide sequence of the secondary precursor sequence of described expression silkworm microRNA is as shown in SEQIDNO.1, SEQIDNO.2 or SEQIDNO.3.
In the present invention, described restructuring pFastBac1 carrier is prepared by following methods: clone's red fluorescent protein encoding gene, then inserts pFastBac1 carrier polyclone enzyme and cuts site, obtain recombinant vectors Red-pFastBac1; The secondary precursor sequence of clonal expression silkworm microRNA again, and then the red fluorescent protein encoding gene downstream of inserting recombinant vectors Red-pFastBac1, obtain the overexpression system of silkworm microRNA.
Preferably, described red fluorescent protein encoding gene inserts EcoRI and the SpeI restriction enzyme site place of pFastBac1 carrier; The secondary precursor sequence of described expression silkworm microRNA inserts XbaI and the KpnI restriction enzyme site place of pFastBac1 carrier.
Beneficial effect of the present invention is: the present invention is by the overexpression system using Bac-to-Bac baculovirus expression system as silkworm microRNA, this expression system can overexpression man small bundle of straw microRNAs, wherein for silkworm let-7 bunch miRNAs, achieve the high expression of foreign gene, overcome the miRNA stand-in of hormone synthetic, cost is high, the phase imitates short defect, overcome the defect that transgene carrier is not suitable for insect cell simultaneously, and provide new instrument for the functional study of silkworm miRNA.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is silkworm pri-let-7, pri-miR-100, pri-miR-2795 and let-7-CPCR amplification (A:pri-let-7 amplification; B:pri-miR-100 amplification; C:pri-miR-2795 amplification; D:let-7-C amplification).
Fig. 2 for restructuring Reporter gene vector builds, (verify, wherein swimming lane 1 ~ 5 represents Rp-let-7 recombinant plasmid, Rp-miR-100 recombinant plasmid, Rp-let-7 recombinant plasmid double digestion, Rp-miR-100 recombinant plasmid double digestion, Red-pFastBac1 plasmid double digestion respectively by A:Rp-let-7, Rp-miR-100 recombinant plasmid and Red-pFastBac1 Reporter gene vector double digestion; B:Rp-miR-2795 recombinant plasmid double digestion is verified, wherein swimming lane 1 and 2 represents Rp-miR-2795 recombinant plasmid, Rp-miR-2795 recombinant plasmid double digestion respectively; C:Rp-let-7-C recombinant plasmid double digestion is verified, wherein swimming lane 1 and 2 represents Rp-let-7-C recombinant plasmid, Rp-let-7-C recombinant plasmid double digestion respectively).
Fig. 3 be the recombination rhabdovirus expression vector of miRNA structure (A:Red-pFastBac1 carrier be connected with goal gene after carrier structure schematic diagram; B: gene structure display after restructuring; PCR detected result after C:Rp-let-7 restructuring; PCR detected result after D:Rp-miR-100E restructuring; PCR detected result after F:RP-miR-2795 restructuring; PCR detected result after A:RP-let-7-C restructuring).
Fig. 4 is Red reporter gene signal detection (A: untreated Sf9 clone (WT) after restructuring baculovirus supernatant infection Sf9 cell; B: negative control AcNPV infects Sf9 clone; C-F: recombinant baculovirus Bac-let-7, Bac-miR-100, Bac-miR-2795 and Bac-let-7-C supernatant liquor infects Sf9 clone (each group is left is white light, and the right side is ruddiness) respectively).
Fig. 5 is that baculovirus miRNA expression vector is at the overexpression of cell levels to miRNAs.
Fig. 6 is that miRNA recombinates Bacmid at the overexpression of individual level to miRNAs.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.
The present invention utilizes baculovirus vector to be an instrument being widely used in Eukaryotic efficiently expressing exogenous gene and recombinant protein, by autographa california nuclear polyhedrosis virus (AcNPV) expression system overexpression silkworm let-7 bunch of miRNAs, the functional study for silkworm miRNA provides new instrument.
The structure of embodiment 1, restructuring Red-FastBac1 Reporter gene vector
Silkworm let-7 (bmo-let-7) is downloaded from miRBase (http://www.mirbase.org/), silkworm miR-100 (bmo-miR-100), silkworm miR-2795 (bmo-miR-2795) secondary precursor sequence, concrete sequence is respectively as SEQIDNO.1, shown in SEQIDNO.2 and SEQIDNO.3, then from domestic silkworm gene group database SilkDB (http://silkworm.swu.edu.cn/cgi-bin/gbrowse/silkdb/), silkworm let-7-C (bmo-let-7-C) full length sequence is obtained, concrete sequence is as shown in SEQIDNO.4, anthozoic Red red fluorescent protein Red gene order is downloaded from ncbi database (http://www.ncbi.nlm.nih.gov/), concrete sequence is as shown in SEQIDNO.5.Then utilize primer5.0 software to design primer pri-let-7, pri-miR-100, pri-miR-2795, let-7-C and Red of amplification bmo-let-7, bmo-miR-100, bmo-miR-2795, bmo-let-7-C and Red respectively, concrete primer is as shown in table 1.
Table 1, pri-let-7, pri-miR-100, pri-miR-2795, let-7-C and Red primer sequence
In table, sequence underscore represents the restriction enzyme site in primer.
With coral polyp cDNA for template, SEQIDNO.14 and SEQIDNO.15 is that primer carries out pcr amplification coral polyp Red gene fragment, then uses EcoRI and SpeI double digestion Red gene fragment, reclaims Red endonuclease bamhi; Cut pFastBac1 plasmid with identical enzyme enzyme simultaneously, reclaim pFastBac1 plasmid backbone, then 1 μ LpFastBac1 plasmid backbone and the 4 μ LRed enzymes of fetching receipts respectively cut back to close fragment, 5h is connected in 16 DEG C with T4 ligase enzyme, obtain recombinant vectors Red-pFastBac1, successively after transforming, choose the checking of spot, bacterium liquid PCR and double digestion, send Shenzhen Hua Da gene sequencing qualification.Result shows that the coral polyp Red gene fragment of cloning is consistent with sequence shown in SEQIDNO.5, shows successful clone coral polyp Red gene.
With rigidifying moth silkworm cDNA for template, to increase respectively the elementary precursor sequence bmo-let-7 of miRNA, bmo-miR-100, bmo-miR-2795 and bmo-let-7-C with primer pri-let-7, pri-miR-100, pri-miR-2795 and let-7-C; 50 μ L reaction systems are 10 × ExTaqbuffer5 μ L, dNTPMixture (2.5mM/Leach) 4 μ L, MgCl 24 μ L, primer each 1 μ L, ExTaq (5U/ μ L) 0.5 μ L, cDNA template 4 μ L, autoclaved distilled water is mended to 50 μ L; Pcr amplification condition: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, Tm anneal 30s, and 72 DEG C extend time, totally 30 circulations; Last 72 DEG C extend 10min; 12 DEG C of preservations.The Tm value of pri-let-7 primer is 58 DEG C, and the extension time is 30s; The Tm value of pri-miR-100 primer is 63 DEG C, and the extension time is 30s; The Tm value of pri-miR-2795 primer is 55 DEG C, and the extension time is 30s; The Tm value of let-7-C primer is 59 DEG C, and the extension time is 3min; The Tm value of Red is 63 DEG C, and the extension time is 50s.Detect pcr amplification product with 1% agarose gel electrophoresis, result as shown in Figure 1.Then glue reclaims object fragment, and be connected to pMD19-Tsimple carrier, obtain bmo-let-7-T, bmo-miR-100-T, bmo-miR-2795-T and let-7-C-T plasmid respectively, after transforming, choose the checking of spot, bacterium liquid PCR and double digestion, send Shenzhen Hua Da gene sequencing qualification.
With XbaI and KpnI double digestion bmo-let-7-T, bmo-miR-100-T and bmo-miR-2795-T plasmid and Red-pFastBac1 carrier, use NotI and XhoI double digestion let-7-C-T plasmid and Red-pFastBac1 carrier simultaneously, and reclaim digestion products (Fig. 2).Then the Red-pFastBac1 carrier framework reclaimed is connected with bmo-let-7, bmo-miR-100, bmo-miR-2795 and let-7-C fragment reclaimed respectively, obtain Red-pFastBac1-let-7 (being called for short Rp-let-7), Red-pFastBac1-miR-100 (being called for short Rp-miR-100), Red-pFastBac1-miR-2795 (being called for short RP-miR-2795) and Red-pFastBac1-let-7-C (being called for short RP-let-7-C), carrier structure is as shown in A in Fig. 3.
Embodiment 2, recombinant baculovirus miRNA Overexpression vector build
Extract Rp-let-7, Rp-miR-100, RP-miR-2795 and RP-let-7-C plasmid, Transformed E .coliDH10Bac/AcNPV competent cell (this laboratory of enlarged culturing preserve containing the genomic intestinal bacteria of AcNPV, according to online DH10Bac competent cell preparation method step preparation), get 100 μ L bacterial suspensions and be coated on containing 7 μ gmL -1gentamicin, 10 μ gmL -1tsiklomitsin, 50 μ gmL -1kantlex, 40 μ gmL -1iPTG and 100 μ gmL -1on the LB plate culture medium of X-gal, 37 DEG C of incubated overnight.Target site mini-attTn7 needed for AcNPV integrates containing pFastBac1 carrier Tn7 transposon, and the helper plasmid of Tn7 transposition protein can be provided containing one in E.coliDH10Bac/AcNPV cell, goal gene can more effectively be recombinated with AcNPV, and after restructuring, gene structure is as shown in B in Fig. 3.And M13 (-40) Forward (being called for short M13-F) and M13Reverse (being called for short M13-R) sequence lay respectively at 128bp and the 145bp place at mini-attTn7 two ends, then bacterium liquid PCR is carried out with primer M13-F/R, verify each recombinant baculovirus DNA, clip size on Red-pFastBac1 carrier between Tn7 is 2430bp, add the length of goal gene, detected result is respectively as shown in C-F in Fig. 3, the recombinant vectors Bacmid of known each miRNA successfully constructs, and confirms further through checking order.
Embodiment 3, restructuring Bacmid transfection Sf9 clone and injection silkworm larva
With each restructuring Bacmid (Bac-miRNA) for test group, the empty viral Bacmid (AcNPV) of non-recombinant is negative control, and the Sf9 cell not adding process is blank (WT); Each restructuring Bacmid, transfection Sf9 cell of 1 μ g is wrapped up with the liposome X-treme of 8 μ L; Change perfect medium after 6-8h is educated in 27 DEG C of cultivations into cultivate; Observe cell at 96h OLYMPUS inverted fluorescence microscope and have stronger red fluorescent protein signal, obtained by the mode of centrifuging and taking supernatant and infect active restructuring Bacmid virus, remove the Sf9 cell infecting fresh culture respectively again, observe extremely strong strong red fluorescent infecting 72h, show that virus breeds (Fig. 4) in a large number, collect s-generation recombinant virus supernatant.Inject each recombinant virus supernatant of the 5 μ L s-generation respectively to silkworm 5 1d in age larva, continuation mulberry leaf raise 5-7d, 5 age 7d round silkworm extract total serum IgE.
Embodiment 4, restructuring Bacmid are at cell levels and individual level miRNAs overexpression
Extract the total serum IgE of Sf9 cell and silkworm with Trizol, 1.2% agarose gel electrophoresis detects RNA quality.Utilize each miRNA of miScriptSYBRGreenKit detection by quantitative of QIAGEN company, establish 3 biology to repeat for each group.QRT-PCR detects let-7, miR-100 and miR-2795 all significantly overexpression found in these 4 test group of Bac-let-7, Bac-miR-100, Bac-miR-2795 and Bac-let-7-C, illustrates that each restructuring Bacmid breeds (Fig. 5) in a large number in Sf9 cell.At cell levels, Bac-let-7-C test group result shows, each member and the equal up-regulated expression of let-7, miR-100 and miR-2795 in bunch, in this coexpression relation and Liu's bodyguard equality report let-7 bunch, the expression pattern of each member is similar, show containing each miRNA precursor in the bmo-let-7-C sequence of cloning, can the ripe body of transcribed miRNA corresponding to processing generation in Sf9 cell.In silkworm individual level, qRT-PCR detected result shows, experiment group B ac-let-7, Bac-miR-100 with Bac-miR-2795 are compared with negative control group (AcNPV), and let-7, miR-100 and miR-2795 be overexpression (in Fig. 6 A-C) all significantly; In Bac-let-7-C group let-7 expression amount and negative control group AcNPV compare no significant difference, but and the expression amount of blank group let-7 there were significant differences (in Fig. 6 D), expression amount remarkable overexpression compared with negative control group of Bac-let-7-C group miR-100, but with blank group there is no notable difference (in Fig. 6 E), Bac-let-7-C group miR-2795 and negative control group are extremely significantly overexpression (in Fig. 6 F) with comparing of blank group.But, after recombinant virus infection silkworm, bmo-let-7, miR-100 and miR-2795 all lower expression (in Fig. 6 A-F) in individuality, this illustrates in silkworm individuality due to a large amount of propagation of virus, silkworm endotrophic is caused to consume in a large number, destroy the eubolism environment of Different Organs in Silkworm, Bombyx mori, tissue and cell, make the expression by inhibitation system of these miRNAs.
Therefore, the present invention build miRNA expression vector, can in silkworm efficiently expressing exogenous gene, the functional study that can be used for silkworm miRNA provides new instrument.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.

Claims (7)

  1. The application of 1.Bac-to-Bac baculovirus expression system in preparation silkworm microRNA overexpression system.
  2. 2. application according to claim 1, it is characterized in that: described silkworm microRNA overexpression system contains restructuring pFastBac1 carrier, described restructuring pFastBac1 carrier is cut site by pFastBac1 carrier polyclone enzyme and is inserted red fluorescent protein encoding gene successively and express the secondary precursor sequence of silkworm microRNA and obtain.
  3. 3. application according to claim 1, it is characterized in that: described restructuring pFastBac1 carrier inserts red fluorescent protein encoding gene by EcoRI and SpeI of pFastBac1 carrier, and inserts the secondary precursor sequence of expression silkworm microRNA in XbaI and KpnI restriction enzyme site and obtain.
  4. 4. the application according to Claims 2 or 3, is characterized in that: the nucleotide sequence of described red fluorescent protein encoding gene is as shown in SEQIDNO.5.
  5. 5. the application according to Claims 2 or 3, is characterized in that: the nucleotide sequence of the secondary precursor sequence of described expression silkworm microRNA is as shown in SEQIDNO.1, SEQIDNO.2 or SEQIDNO.3.
  6. 6. the application described in any one of claims 1 to 3, it is characterized in that: described restructuring pFastBac1 carrier is prepared by following methods: clone's red fluorescent protein encoding gene, then insert pFastBac1 carrier polyclone enzyme and cut site, obtain recombinant vectors Red-pFastBac1; The secondary precursor sequence of clonal expression silkworm microRNA again, and then the red fluorescent protein encoding gene downstream of inserting recombinant vectors Red-pFastBac1, obtain the overexpression system of silkworm microRNA.
  7. 7. application according to claim 6, is characterized in that: described red fluorescent protein encoding gene inserts EcoRI and the SpeI restriction enzyme site place of pFastBac1 carrier; The secondary precursor sequence of described expression silkworm microRNA inserts XbaI and the KpnI restriction enzyme site place of pFastBac1 carrier.
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN106636208A (en) * 2016-11-10 2017-05-10 西南大学 Method of utilizing bombyx mori nuclear polyhedrosis virus vector system to excessively express microRNA

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CN106636208A (en) * 2016-11-10 2017-05-10 西南大学 Method of utilizing bombyx mori nuclear polyhedrosis virus vector system to excessively express microRNA

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