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

Abstract

The invention discloses the application of the Bac-to-Bac baculovirus expression system in the preparation of the silkworm microRNA overexpression system. The silkworm microRNA overexpression system contains a recombinant pFastBac1 vector, and the recombinant pFastBac1 vector is obtained from the multiple cloning enzyme cutting site of the pFastBac1 vector. It is obtained by sequentially inserting the red fluorescent protein coding gene and the secondary precursor sequence expressing silkworm microRNA. The present invention utilizes the Bac-to-Bac baculovirus expression system as the silkworm microRNA overexpression system to realize superexpression at both the silkworm cell and individual levels. Quantitative expression of silkworm cluster microRNAs provides a new tool for the functional study of silkworm miRNAs.

Description

Application of Bac-to-Bac Baculovirus Expression System in Preparation of Bombyx mori microRNA Overexpression System

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of a Bac-to-Bac baculovirus expression system in preparing a silkworm microRNA overexpression system.

Background technique

The silkworm (Bombyxmori) is a Lepidoptera insect with important economic value and scientific research value. With the analysis of the silkworm genome, the research on silkworm has entered the era of functional genome. miRNA technology is one of the commonly used technologies for functional gene research. At present, the common method of miRNA is to inject artificially synthesized miRNA mimics in vitro or to express silkworm miRNA efficiently in vivo through transgenic vectors, but injecting artificially synthesized miRNA mimics in vitro is costly. , short-term effect; however, the high-efficiency expression of silkworm miRNA through transgenic vectors is limited by the expression vectors. The reason is that most commercial miRNA expression vectors are suitable for mammalian cells, but not suitable for the expression of miRNA in insect cells. .

Therefore, there is an urgent need for a miRNA expression system suitable for silkworms, which can efficiently express foreign genes in silkworm individuals or cells, and provides a new tool for the functional research of silkworm miRNAs.

Contents of the invention

In view of this, one of the objectives of the present invention is to provide the application of Bac-to-Bac baculovirus expression system in the preparation of Bombyx mori microRNA overexpression system.

In order to realize the foregoing invention object, the present invention provides following technical scheme:

Application of Bac-to-Bac baculovirus expression system in preparation of silkworm microRNA overexpression system.

In the present invention, the silkworm microRNA overexpression system contains a recombinant pFastBac1 vector, and the recombinant pFastBac1 vector is formed by sequentially inserting a red fluorescent protein encoding gene and a secondary precursor sequence expressing silkworm microRNA at the pFastBac1 vector multiple cloning restriction site. have to.

Preferably, the recombinant pFastBac1 vector is obtained by inserting the red fluorescent protein coding gene at EcoRI and SpeI of the pFastBac1 vector, and inserting the secondary precursor sequence expressing silkworm microRNA at the XbaI and KpnI restriction sites.

More preferably, the nucleotide sequence of the red fluorescent protein encoding gene is shown in SEQ ID NO.5.

More preferably, the nucleotide sequence of the secondary precursor sequence expressing silkworm microRNA is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

In the present invention, the recombinant pFastBac1 vector is prepared by the following method: cloning the red fluorescent protein coding gene, and then inserting it into the multi-cloning restriction site of the pFastBac1 vector to obtain the recombinant vector Red-pFastBac1; and then cloning the secondary precursor expressing silkworm microRNA sequence, and then inserted into the downstream of the red fluorescent protein coding gene of the recombinant vector Red-pFastBac1 to obtain the overexpression system of silkworm microRNA.

Preferably, the red fluorescent protein encoding gene is inserted into the EcoRI and SpeI restriction sites of the pFastBac1 vector; the secondary precursor sequence expressing the silkworm microRNA is inserted into the XbaI and KpnI restriction sites of the pFastBac1 vector.

The beneficial effects of the present invention are: the present invention uses the Bac-to-Bac baculovirus expression system as the overexpression system of silkworm microRNA, and the expression system can overexpress silkworm cluster microRNAs, wherein the silkworm let-7 cluster miRNAs are used as the overexpression system. For example, the high-efficiency expression of exogenous genes has been achieved, and the defects of high cost and short-term effect of injecting artificially synthesized miRNA mimics in vitro have been overcome. At the same time, the defect that transgenic vectors are not suitable for insect cells has been overcome, and the function of silkworm miRNA has been overcome. Research provides new tools.

Description of drawings

In order to make the purpose, technical scheme and beneficial effect of the present invention clearer, the present invention provides the following drawings:

Figure 1 shows the amplification results of silkworm pri-let-7, pri-miR-100, pri-miR-2795 and let-7-CPCR (A: amplification result of pri-let-7; B: amplification result of pri-miR-100 Amplification result; C: pri-miR-2795 amplification result; D: let-7-C amplification result).

Figure 2 shows the construction of the recombinant reporter gene vector (A: Rp-let-7, Rp-miR-100 recombinant plasmid and Red-pFastBac1 reporter gene vector double enzyme digestion verification, in which lanes 1 to 5 represent the Rp-let-7 recombinant plasmid , Rp-miR-100 recombinant plasmid, Rp-let-7 recombinant plasmid double enzyme digestion, Rp-miR-100 recombinant plasmid double enzyme digestion, Red-pFastBac1 plasmid double enzyme digestion; B: Rp-miR-2795 recombinant plasmid double enzyme Cutting verification, in which lanes 1 and 2 represent Rp-miR-2795 recombinant plasmid, Rp-miR-2795 recombinant plasmid double enzyme digestion; C: Rp-let-7-C recombinant plasmid double restriction digestion verification, in which lanes 1 and 2 Respectively represent Rp-let-7-C recombinant plasmid, Rp-let-7-C recombinant plasmid (double digestion).

Fig. 3 is the construction of the recombinant baculovirus expression vector of miRNA (A: Red-pFastBac1 carrier and target gene connection after vector structure diagram; B: gene structure diagram after recombination; C: PCR detection result after Rp-let-7 recombination; D: PCR detection results after Rp-miR-100E recombination; F: PCR detection results after RP-miR-2795 recombination; A: PCR detection results after RP-let-7-C recombination).

Figure 4 is the detection of Red reporter gene signal after recombinant baculovirus supernatant infected Sf9 cells (A: untreated Sf9 cell line (WT); B: negative control AcNPV infected Sf9 cell line; C-F: recombinant baculovirus Bac- The supernatants of let-7, Bac-miR-100, Bac-miR-2795 and Bac-let-7-C were infected with Sf9 cell line respectively (the left of each group is white light, the right is red light)).

Figure 5 shows the overexpression of miRNAs by the baculovirus miRNA expression vector at the cellular level.

Figure 6 shows the overexpression of miRNAs by miRNA recombinant Bacmid at the individual level.

detailed description

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. For the experimental methods not specified in the examples, the conventional conditions are generally followed, such as the conditions described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al.), or the conditions suggested by the manufacturer.

The present invention uses the baculovirus vector, which is a tool widely used in eukaryotes to efficiently express foreign genes and recombinant proteins, to overexpress the Bombyx mori let- Seven clusters of miRNAs provide a new tool for the functional study of silkworm miRNAs.

Embodiment 1, the construction of recombinant Red-FastBac1 reporter gene carrier

Download silkworm let-7 (bmo-let-7), silkworm miR-100 (bmo-miR-100), silkworm miR-2795 (bmo-miR-2795) from miRBase (http://www.mirbase.org/) The secondary precursor sequence, the specific sequence is shown in SEQIDNO.1, SEQIDNO.2 and SEQIDNO.3 respectively, and then from silkworm genome database SilkDB (http://silkworm.swu.edu.cn/cgi-bin/gbrowse/silkdb /) to obtain the full-length sequence of Bombyx mori let-7-C (bmo-let-7-C), the specific sequence is shown in SEQ ID NO.4, from the NCBI database (http://www.ncbi.nlm.nih.gov/ ) to download the Red gene sequence of red fluorescent protein Red of coral polyps, the specific sequence is shown in SEQ ID NO.5. Then use primer5.0 software to design primers pri-let-7, pri-miR-100 for amplifying bmo-let-7, bmo-miR-100, bmo-miR-2795, bmo-let-7-C and Red respectively , pri-miR-2795, let-7-C and Red, the specific primers are shown in Table 1.

Table 1, pri-let-7, pri-miR-100, pri-miR-2795, let-7-C and Red primer sequences

The underlined sequence in the table indicates the enzyme cleavage site in the primer.

Use coral polyp cDNA as a template, and use SEQIDNO.14 and SEQIDNO.15 as primers to amplify the coral polyp Red gene fragment by PCR, then use EcoRI and SpeI to double-digest the Red gene fragment, and recover the Red enzyme-digested fragment; at the same time, use the same enzyme to digest pFastBac1 Plasmid, recover the pFastBac1 plasmid backbone, then take the recovered 1 μL pFastBac1 plasmid backbone and 4 μL Red enzyme-digested recovery fragments, and use T4 ligase at 16°C for 5 hours to obtain the recombinant vector Red-pFastBac1, which is transformed, spot-picked, bacterial liquid PCR and After double-enzyme digestion verification, it will be sent to Shenzhen Huada Gene for sequencing identification. The results showed that the cloned polyp Red gene fragment was consistent with the sequence shown in SEQ ID NO.5, indicating that the polyp Red gene was successfully cloned.

Using the cDNA of the rigid moth Bombyx mori as a template, primers pri-let-7, pri-miR-100, pri-miR-2795 and let-7-C were used to amplify the primary miRNA precursor sequences bmo-let-7, bmo- miR-100, bmo-miR-2795 and bmo-let-7-C; 50μL reaction system: 10×ExTaqbuffer 5μL, dNTPMixture (2.5mM/Leach) 4μL, MgCl ₂ 4μL, each primer 1μL, ExTaq (5U/μL) 0.5 μL, 4 μL of cDNA template, made up to 50 μL with autoclaved double distilled water; PCR amplification conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, Tm annealing for 30 s, extension time at 72°C, a total of 30 cycles; final 72°C Extend for 10 min; store at 12°C. The Tm value of pri-let-7 primer was 58℃, and the extension time was 30s; the Tm value of pri-miR-100 primer was 63℃, and the extension time was 30s; the Tm value of pri-miR-2795 primer was 55℃, and the extension time The time is 30s; the Tm value of let-7-C primer is 59°C, and the extension time is 3min; the Tm value of Red is 63°C, and the extension time is 50s. The PCR amplification products were detected by 1% agarose gel electrophoresis, and the results are shown in Figure 1. Then the target fragment was recovered by gel and connected to the pMD19-Tsimple vector to obtain bmo-let-7-T, bmo-miR-100-T, bmo-miR-2795-T and let-7-CT plasmids respectively. After transformation, Spot-picking, bacterial liquid PCR and double enzyme digestion were verified and then sent to Shenzhen Huada Genomics for sequencing identification.

Double-digest bmo-let-7-T, bmo-miR-100-T and bmo-miR-2795-T plasmids and Red-pFastBac1 vector with XbaI and KpnI, and double-digest let-7-C-T with NotI and XhoI Plasmids and Red-pFastBac1 vectors, and the digested products were recovered (Figure 2). Then the recovered Red-pFastBac1 vector backbone was connected with recovered bmo-let-7, bmo-miR-100, bmo-miR-2795 and let-7-C fragments respectively to obtain Red-pFastBac1-let-7 (referred to as Rp -let-7), Red-pFastBac1-miR-100 (referred to as Rp-miR-100), Red-pFastBac1-miR-2795 (referred to as RP-miR-2795) and Red-pFastBac1-let-7-C (referred to as RP -let-7-C), the carrier structure is shown in A in Figure 3.

Embodiment 2, recombinant baculovirus miRNA overexpression vector construction

Extract Rp-let-7, Rp-miR-100, RP-miR-2795 and RP-let-7-C plasmids, transform E.coliDH10Bac/AcNPV competent cells (expand the large intestine containing AcNPV genome preserved in our laboratory Bacillus, prepared according to the preparation method of DH10Bac competent cells on the Internet), take 100 μL of bacterial suspension and spread it in the solution containing 7 μg·mL ^-1 gentamicin, 10 μg·mL ^-1 tetracycline, 50 μg·mL ^-1 kanamycin, 40 μg·mL ^-1 IPTG and 100 μg·mL ^-1 X-gal on LB plate medium, culture overnight at 37°C. Since AcNPV contains the target site mini-attTn7 required for the integration of pFastBac1 vector transposon Tn7, and E.coliDH10Bac/AcNPV cells contain a helper plasmid that can provide Tn7 transposable protein, so that the target gene can be more effectively integrated with AcNPV Recombination occurs, and the gene structure after recombination is shown in B in FIG. 3 . And the sequences of M13(-40)Forward (M13-F for short) and M13Reverse (M13-R for short) are located at 128bp and 145bp at both ends of mini-attTn7, respectively, and then use primer M13-F/R to carry out bacterial liquid PCR to verify that each recombinant Baculovirus DNA, the size of the fragment between Tn7 on the Red-pFastBac1 vector is 2430bp, plus the length of the target gene, the test results are shown in Fig. further confirm.

Embodiment 3, recombinant Bacmid transfection Sf9 cell line and injection silkworm larvae

Each recombinant Bacmid (Bac-miRNA) was used as the test group, non-recombinant empty virus Bacmid (AcNPV) was used as the negative control, and untreated Sf9 cells were used as the blank control (WT); Each recombinant Bacmid was transfected into Sf9 cells; cultured at 27°C for 6-8 hours and then replaced with complete medium; at 96 hours, it was observed that the cells had a strong red fluorescent protein signal with an OLYMPUS inverted fluorescence microscope, and the supernatant was obtained by centrifugation Obtain recombinant Bacmid virus with infective activity, and then infect freshly cultured Sf9 cells. A very strong red fluorescent signal was observed at 72 hours after infection, indicating that the virus had proliferated in large quantities (Figure 4). Collect the second-generation recombinant virus on the clear. Inject 5 μL of the supernatant of each second-generation recombinant virus to the 5th instar 1d larvae of silkworm, and continue to feed them with mulberry leaves for 5-7d, and extract the total RNA from the whole silkworm at 5th instar 7d.

Example 4, Recombinant Bacmid Overexpression of miRNAs at Cellular Level and Individual Level

The total RNA of Sf9 cells and silkworm was extracted with Trizol, and the quality of RNA was detected by 1.2% agarose gel electrophoresis. The miScriptSYBRGreenKit from QIAGEN was used to quantitatively detect each miRNA, and three biological replicates were set up for each group. qRT-PCR detection found that let-7, miR-100 and miR-2795 in the four test groups of Bac-let-7, Bac-miR-100, Bac-miR-2795 and Bac-let-7-C were significantly Overexpression indicated that each recombinant Bacmid had proliferated in Sf9 cells in large quantities (Fig. 5). At the cellular level, the results of the Bac-let-7-C test group showed that each member of the cluster, namely let-7, miR-100 and miR-2795, was up-regulated. This co-expression relationship is consistent with the let-7 The expression patterns of each member in the cluster were similar, indicating that the cloned bmo-let-7-C sequence contained various miRNA precursors, which could be transcribed and processed into corresponding mature miRNAs in Sf9 cells. At the silkworm individual level, the results of qRT-PCR detection showed that compared with the negative control group (AcNPV), Bac-let-7, Bac-miR-100 and Bac-miR-2795 in the experimental group, let-7, miR-100 and miR -2795 was significantly overexpressed (A-C in Figure 6); the expression of let-7 in the Bac-let-7-C group was not significantly different from that of the negative control group AcNPV, but it was different from the expression of let-7 in the blank control group There was a significant difference in the amount of miR-100 (D in Figure 6), and the expression of miR-100 in the Bac-let-7-C group was significantly overexpressed compared with that of the negative control group, but there was no significant difference with that of the blank control group (E in Figure 6 ), miR-2795 in the Bac-let-7-C group was extremely significantly overexpressed compared with the negative control group and the blank control group (Fig. 6, F). However, after the recombinant virus infected the silkworm, the expression of bmo-let-7, miR-100 and miR-2795 were all down-regulated in the individual (Fig. A large amount of nutrients are consumed, which destroys the normal metabolic environment of different organs, tissues and cells in the silkworm, and inhibits the expression of these miRNAs.

Therefore, the miRNA expression vector constructed by the present invention can efficiently express exogenous genes in silkworm, and can be used for the functional research of silkworm miRNA to provide a new tool.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that it can be described in terms of form and Various changes may be made in the details without departing from the scope of the invention defined by the claims.

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.