CN116478262B - Application of RACK1 in resisting silkworm nuclear polyhedrosis virus BmNPV - Google Patents
Application of RACK1 in resisting silkworm nuclear polyhedrosis virus BmNPV Download PDFInfo
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Abstract
The invention discloses an application of RACK1 in resisting silkworm nuclear polyhedrosis virus BmNPV, wherein the amino acid sequence of RACK1 is shown as SEQ ID NO.6, and research shows that RACK1 is specifically combined with BmST; the proliferation of BmNPV can be obviously inhibited by the increment expression RACK1; therefore, RACK1 has important value in research and application of silkworm antiviral molecular breeding, can improve the antiviral capability of silkworms by transferring and increasing expression of the gene, can also be used as a proliferation inhibitor of the silkworm nuclear polyhedrosis virus BmNPV, and has important significance for resisting the silkworm nuclear polyhedrosis virus BmNPV.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to an application of RACK1 in resisting silkworm nuclear polyhedrosis virus BmNPV.
Background
Silkworm is an important economic insect, and silk is an important raw material for silk industry. In developing countries such as China, india, brazil, etc., the silkworm industry is an important source of economic income for farmers, even in some areas. However, silkworm is faced with serious disease threat, and nuclear polyhedrosis virus disease is the most common and serious type of silkworm disease in silkworm industry production, and the pathogen causing the disease is nuclear polyhedrosis virus (BmNPV), and the disease has extremely strong infectious power and is difficult to control.
Since BmNPV is serious in silkworm production, scientific researchers have been hoped to find out key genes affecting the resistance of silkworms to viruses, and then to improve the resistance of silkworms to viruses through molecular biology technology. The cloning and identification of the full-length sequence of the key gene affecting the resistance of silkworm virus has important theoretical value and practical significance for elucidating the mechanism of resisting silkworm virus and cultivating resistant varieties for silkworm production.
The chinese patent with publication number CN106834298A identifies the silkworm resistance key gene BmST, but how this gene functions is not clear at present, whether there are other antiviral genes. Therefore, identification of other silkworm resistance key genes is needed, and the method has important significance for breeding resistant varieties.
Disclosure of Invention
In view of the above, the present invention aims to provide a full-length sequence of a key gene RACK1 derived from silkworms for controlling the resistance of silkworms to viruses such as BmNPV, and the like, carrying out prokaryotic expression on BmST, purifying recombinant protein GST-BmST, screening candidate proteins RACK1 interacting with BmST through pull down and mass spectrometry, and proving that RACK1 is combined with BmST through pull down and Co-IP, wherein the proliferation of BmNPV can be obviously inhibited by the cell level up-expression of RACK1, which indicates that the gene is a key gene affecting the resistance of silkworms to viruses, and can be used as a proliferation inhibitor of the silkworm nuclear polyhedrosis virus BmNPV.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the application of RACK1 in preparing proliferation inhibitors of silkworm nuclear polyhedrosis virus BmNPV, wherein the amino acid sequence of RACK1 is shown as SEQ ID NO. 6.
Preferably, the nucleotide sequence of RACK1 is shown as SEQ ID NO. 5.
2. The application of the increased expression RACK1 in preparing a transformant for resisting silkworm nuclear polyhedrosis virus BmNPV, wherein the amino acid sequence of the RACK1 is shown as SEQ ID NO. 6.
Preferably, the nucleotide sequence of RACK1 is shown as SEQ ID NO. 5.
Preferably, the method for expressing RACK1 in an increasing mode comprises the steps of constructing a recombinant vector for expressing RACK1 genes, and then transforming the recombinant vector into a receptor cell to obtain a transformant.
Preferably, the RACK1 gene in the recombinant vector is regulated and controlled to be expressed by a silkworm Actin 4 promoter and an SV40 termination signal sequence.
Preferably, the transformant is a silkworm cell or a silkworm individual.
The invention has the beneficial effects that: according to the invention, protein expression purification, pull down and mass spectrometry analysis are carried out on the resistance gene BmST, so that the candidate protein RACK1 for interaction is screened; the specific combination of RACK1 and BmST is proved by in vitro pull down and in vivo Co-IP experiments; the proliferation of BmNPV can be obviously inhibited by the increased expression of RACK1. Therefore, RACK1 has important value in research and application of silkworm antiviral molecular breeding, can improve the antiviral capability of silkworms by transferring and increasing expression of the gene, can also be used as a proliferation inhibitor of the silkworm nuclear polyhedrosis virus BmNPV, and has important significance for resisting the silkworm nuclear polyhedrosis virus BmNPV.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 shows the expression and purification of BmST protein and the pull down detection (A: purified GST-BmST-His recombinant protein; B: bmNPV, bmNPV-infected midgut and BmNPV-infected BmE cell total protein, respectively as input samples for pull down detection by electrophoresis silver staining, two specific binding bands (red arrow) were present in BmNPV-infected cell samples, and mass spectrometry was performed by gel digging to screen candidate interacting proteins).
FIG. 2 shows RACK1-Flag transfected cells as input, and pull down binding experiments with GST-BmST-His and GST, followed by WB detection using Flag, GST, his antibody.
FIG. 3 shows GST-RACK1-Flag protein purification and pull down binding experiments (A: purified GST-RACK1-Flag recombinant protein; B: bmST-His transfected cells as input, pull down binding experiments with GST-RACK1-Flag and GST, followed by WB detection using Flag, GST, his antibody).
FIG. 4 shows the Co-IP binding assay of RACK1 with BmST (expression plasmids of RACK1-flag+BmST-His and EGFP-flag+BmST-His were transfected into BmE cells, respectively, total cell proteins were collected as inputs, immunoprecipitation (IP) was performed using Flag and His antibodies, respectively, and then samples after input and IP were subjected to WB detection using Flag and His antibodies, respectively).
FIG. 5 shows that the increased expression of RACK1 gene in BmE cells can significantly inhibit BmNPV proliferation (A: RACK1 gene expression level is detected by 24h PCR after transfection, protein content is detected by 48h WB, bmNPV virus content is detected by 48h qPCR after virus infection, and BmNPV virus fluorescence is observed by 72h after virus infection).
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
According to the gene sequence of BmST, designing a primer, cloning BmST gene (BmST-His) with His tag, then adding the BmST gene to prokaryotic expression pGEX-6p-1 with GST tag, and obtaining GST-BmST-His protein through induced expression and protein purification. Extracting total proteins of silkworm cells and individuals infected with viruses, carrying out pull down detection on the total proteins and GST-BmST-His, cutting specific binding bands for mass spectrometry after electrophoresis detection, and screening interaction candidate protein RACK1 from the top 20 proteins in a mass spectrometry identification result. The RACK1 gene with Flag (RACK 1-Flag) is cloned, a transient increment expression vector 1180-Hr3-A4P-RACK1-Flag-SV40 (RACK 1 OE) is constructed, cells are transfected, total protein is collected after cell extraction and is used as an input sample, and the input sample is subjected to pull down detection with GST-BmST-His, and then Western Blot (WB) detection is carried out by using a Flag antibody. The prokaryotic expression vector of RACK1-Flag is constructed, recombinant protein GST-RACK1-Flag is purified, the cell increment expression vector 1180-Hr3-A4P-BmST-His-SV40 (BmSTOE) of BmST-His is constructed, total protein is extracted as input samples after transfected cells, and pull down analysis and WB detection are carried out with GST-RACK 1-Flag. 1180-Hr3-A4P-EGFP-Flag-SV40 (EGFPOE) is used as a control of RACK1OE, RACK1OE and BmSTOE cotransverse cells, EGFPOE and BmSTOE cotransverse are used as a control, co-IP experiments are respectively carried out by using Flag and His antibodies, and then WB detection is carried out. RACK1OE and 1180 control transfected cells were used for RT-PCR detection of RACK1 after 24h RNA extraction, flag tags were detected by 48h protein extraction, bmNPV was infected 48h after transfection, viral DNA content was detected 48h after infection and viral fluorescence was observed 72 h. The in vivo and in vitro detection results show that RACK1 is specifically combined with BmST to inhibit proliferation of BmNPV, and the RACK1 is a key gene affecting the virus resistance of silkworms and can be used as a breeding target gene of antiviral molecules of the silkworms.
Example 1 purification of expression of BmST protein and Pull Down detection
First, a primer with His tag is designed according to the cloned BmST sequence:
BmST-HisF: 5'-GGATCCATGCACCGCCATTTTCTTT-3' (SEQ ID NO. 1); bmST-HisR: 5'-GCGGCCGCTCAATGGTGATGGTGATGATGAACAAAGATGAAGTCAGGGT-3' (SEQ ID NO. 2), amplification was performed using the stored BmST plasmid as a template, and the PCR reaction conditions were: pre-denaturation at 94℃for 4 min, then denaturation at 94℃for 40 sec, annealing at 58℃for 40 sec, extension at 72℃for 40 sec, 28 cycles total, and finally extension at 72℃for 10 min; the PCR product is identified and recovered by agarose gel electrophoresis, then is connected with a pMD19-T vector, the connection reaction is carried out under the action of T4 DNA ligase, the connection reaction is carried out at 16 ℃ overnight, DH5 alpha competent cells are transformed, positive clones are obtained and then are sent to Shanghai Biotechnology limited company for sequencing, and the sequencing result shows that BmST-His genes with His labels are successfully cloned.
Carrying out double digestion on the plasmid successfully sequenced in the previous step by BamH I and Not I, and carrying out agarose electrophoresis identification and recovery to obtain BmST-His digestion fragments; meanwhile, bamH I and Not I are used for double digestion of a prokaryotic expression vector pGEX-6p-1 with GST tag, bmST-His enzyme digestion fragments and pGEX-6p-1 enzyme digestion fragments are connected and transformed, positive clones are screened, and pGEX-6p-1-BmST-His vector is obtained.
pGEX-6p-1-BmST-His plasmid is transferred into BL21 competent cells, plating is carried out overnight, monoclonal is selected for culture, different concentrations of IPTG are added for induction expression under the conditions of different temperatures (16 ℃, 25 ℃ and 37 ℃), and electrophoresis detection results show that 0.4% of IPTG is used for induction expression for 20 hours at 16 ℃, and the GST-BmST-His of the target protein is obvious in the supernatant.
And (3) carrying out mass induction expression according to the conditions, collecting bacterial liquid, centrifuging, carrying out ultrasonic crushing after re-suspending, collecting supernatant after centrifuging, purifying by using a GST affinity chromatography column, eluting by using reduced glutathione, and desalting to obtain the GST-BmST-His recombinant protein (A in figure 1).
Extracting total proteins of midgut and BmE after BmNPV infection for 48h and 72h respectively, and performing pull down detection with GST-BmST-His recombinant protein respectively, and performing silver staining detection after electrophoresis, wherein the results are shown in figure 1B. The results showed that in the BmNPV-infected cell samples there were two specific binding bands, which were gummed for mass spectrometry.
The mass spectrum identifies 422 specific peptide fragments, reliability ranking is carried out according to protein coverage, and the proteins of 20 ranked top are respectively analyzed to screen out candidate protein RACK1.
Example 2, RACK1 and BmST pull Down in vitro binding experiments
(1) Firstly, designing a specific primer according to the genome sequence of the silkworm:
RACK 1F: 5'-GGATCCATGTCTGAAACATTAAAACTCC-3' (SEQ ID NO. 3); RACK 1R: 5'-GCGGCCGCTTATCGAGCTGAGATGGAAAC-3' (SEQ ID NO. 4), the whole silkworm cDNA of 5 th year and 3 rd day of silkworm is used as a template for amplification, and the PCR reaction conditions are as follows: pre-denaturation at 94℃for 4 min, then denaturation at 94℃for 40 sec, annealing at 55℃for 40 sec, extension at 72℃for 50 sec for a total of 30 cycles, and finally extension at 72℃for 10 min; the PCR product is identified and recovered by agarose gel electrophoresis and then is connected with a pMD19-T carrier, the connection reaction is carried out under the action of T4 DNA ligase, the connection reaction is carried out at 16 ℃ overnight, DH5 alpha competent cells are transformed, positive clones are obtained and then are sent to Shanghai Biotechnology limited company for sequencing, the sequencing result shows that the CDS sequence of RACK1 gene is successfully cloned, the full length is 960bp, the nucleotide sequence is shown as SEQ ID NO.5, and the coded amino acid is shown as SEQ ID NO. 6.
(2) Taking the plasmid successfully sequenced in the previous step as a template, and using a specific primer RACK1-Flag F:5' -GGAT CCATGGATTACAAGGATGACGACGATAAGTCTGAAACATTAAAACTCC-3’(SEQ ID NO.7)；
RACK1-Flag R：5’-GCGGCCGCTTACTTATCGTCGTCATCCTTGTAATCTCGAGCTGAGATGGAAAC-3' (SEQ ID NO. 8) is amplified, the target fragment is recovered and then connected with a pMD19-T vector, and spot picking sequencing is transformed to obtain the RACK1-Flag gene with Flag label.
(3) Double enzyme cutting is carried out on RACK1-Flag gene plasmid by BamH I and Not I, agarose electrophoresis identification is carried out, and recovery is carried out, thus obtaining RACK1-Flag enzyme cutting fragments; meanwhile, the vector 1180 which already contains the Hr3 enhancer, the silkworm Actin 4 promoter (A4P) and the SV40 termination signal sequence is subjected to double digestion by BamH I and Not I, the hycu-ep32 gene sequence of the pb-HEAG vector is replaced by RACK1-Flag (the pb-HEAG vector is seen in Jiang Liangbo Pi paper, research on resistance materials and resistance mechanisms of silkworm nuclear polyhedrosis virus (BmNPV) based on transgenic engineering, 2013), and the 1180-Hr3-A4P-SV40 digestion fragment is obtained through agarose electrophoresis identification and recovery. And (3) performing ligation transformation on the RACK1-Flag enzyme fragments and 1180-hr3-A4P-SV40 enzyme fragments, and screening positive clones to obtain 1180-hr3-A4P-RACK1-Flag-SV40 vector (RACK 1OE for short).
(4) RACK1OE was transfected into BmE cells, and after 48 hours, total protein was collected from the cells and used as input samples, and pull down experiments were performed with GST-BmST-His and GST proteins, respectively, and WB detection was performed using Flag, GST, his antibody, and the results are shown in FIG. 2. The results showed that RACK1 binds to GST-BmST-His and not to GST.
(5) And (3) carrying out double enzyme digestion on the RACK1-Flag gene plasmid and the pGEX-6p-1 vector respectively by using BamH I and Not I, recovering RACK1-Flag enzyme fragments and pGEX-6p-1 enzyme fragments, carrying out connection transformation, and screening positive clones to obtain the pGEX-6p-1-RACK1-Flag vector. The GST-RACK1-Flag recombinant protein was obtained by purification with reference to the expression and purification method of GST-BmST-His recombinant protein (FIG. 3A).
(6) The BmST-His fragment obtained by double digestion of BamHI and Not I and 1180-hr3-A4P-SV40 fragment were subjected to ligation transformation, and positive clones were selected to obtain 1180-hr3-A4P-BmST-His-SV40 vector (BmSTOE). Total cell proteins obtained after BmSTOE transfection were used as inputs for pull down experiments with GST-RACK1-Flag and GST proteins, respectively, and WB detection was performed using Flag, GST, his antibody, and the results are shown in FIG. 3B. The results showed that BmST bound to GST-RACK1-Flag and not GST.
(7) The above forward and reverse in vitro pull down results indicate that RACK1 specifically binds to BmST.
Co-IP in vivo binding experiments for RACK1 and BmST example 3
The Flag-tagged EGEP expression plasmid 1180-hr3-A4P-EGFP-Flag-SV40 (EGFPOE) stored in the laboratory was used as a control for RACK1OE, RACK1OE+BmSTOE and EGFPOE+BmSTOE were transfected into BmE cells, respectively, and total protein was collected from the cells as an input sample.
(2) The two groups of input samples were Immunoprecipitated (IP) with Flag and His antibodies, respectively, and then the samples after input and IP were WB detected with Flag and His antibodies, respectively, and the results are shown in FIG. 4. The results show that BmST binds to RACK1 and not EGFP.
(3) Combining the in vitro pull down and in vivo Co-IP results, RACK1 and BmST are proved to be specifically combined.
Example 4 inhibition of BmNPV proliferation by increased expression of RACK1
(1) RACK1OE and control 1180 plasmids (pSL 1180 plasmid) were transfected into BmE cells, respectively, RNA was extracted 24h after transfection and reverse transcribed into cDNA, using RACK1 gene specific primers:
(2) RACK1qRT F:5'-CAACTAATCCGAAATACCCG-3' (SEQ ID NO. 9); RACK1qRT R:5'-CAGAAATGAAGTGCGAATGAC-3' (SEQ ID NO. 10) and the specific primer TIF-4AqRT F for the internal reference gene TIF-4A: 5'-GAATGGACCCTGGGACACTT-3' (SEQ ID NO. 11), TIF-4AqRT R:5'-CTGACTGGGCTTGAGCGATA-3' (SEQ ID NO. 12) was subjected to RT-PCR detection, and the results are shown in FIG. 5A. The results showed that the amplified band brightness of RACK1 was more pronounced in the RACK1OE transfected samples. Total cell proteins were extracted 48h after transfection, WB detection was performed using Flag-tagged antibodies, GAPDH antibodies were used as controls, and the results are shown in FIG. 5A. The results show that a specific band was detected in the RACK1OE transfected samples. These results indicate that RACK1 upregulation was successful.
(2) Infection with BmNPV-GFP virus with green fluorescent label 48h after transfection, DNA extraction 48h after infection with BmNPV virus GP41 gene specific primer GP41qRT F:5'-CGTAGTAGTAGTAATCGCCGC-3' (SEQ ID NO. 13), GP41qRT R:5'-AGTCGAGTCGCGTCGCTTT-3' (SEQ ID NO. 14) by qPCR detection, the silkworm housekeeping gene BmGAPDH is taken as an internal reference, and a specific detection primer is BmGAPDHqRT F:5'-CCGCGTCCCTGTTGCTAAT-3' (SEQ ID NO. 15), bmGAPDHqRT R:5'-CTGCCTCCTTGACCTTTTGC-3' (SEQ ID NO. 16) was carried out according to the instructions of the instrument and kit, the results being shown in FIG. 5B. qPCR detection results show that cells transfected with RACK1OE can significantly inhibit virus proliferation, and BmNPV content is only 38% of that of a control.
(3) Fluorescence was observed 72h after infection with virus, and the results are shown in fig. 5C. The results showed that the virus fluorescence in cells transfected with RACK1OE was significantly less than that of the control, indicating that increased expression of RACK1 was able to inhibit proliferation of BmNPV.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (6)
- The application of RACK1 in preparing proliferation inhibitors of silkworm nuclear polyhedrosis virus BmNPV is characterized in that: the amino acid sequence of RACK1 is shown as SEQ ID NO. 6.
- 2. The use according to claim 1, characterized in that: the nucleotide sequence of RACK1 is shown as SEQ ID NO. 5.
- 3. The application of the increased expression RACK1 in preparing transformants resistant to silkworm nuclear polyhedrosis virus BmNPV is characterized in that: the amino acid sequence of RACK1 is shown as SEQ ID NO. 6; the transformant is a silkworm cell or a silkworm individual.
- 4. A use according to claim 3, characterized in that: the nucleotide sequence of RACK1 is shown as SEQ ID NO. 5.
- 5. A use according to claim 3, characterized in that: the method for expressing RACK1 in an increasing way is to construct a recombinant vector for expressing RACK1 genes, and then to transform the recombinant vector into receptor cells to obtain transformants.
- 6. The use according to claim 5, characterized in that: the RACK1 gene in the recombinant vector is expressed under the regulation of a silkworm Actin 4 promoter and an SV40 termination signal sequence.
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| CN106834298A (en) * | 2017-01-17 | 2017-06-13 | 西南大学 | Silkworm BmST genes and its application |
| CN111534544A (en) * | 2020-05-07 | 2020-08-14 | 西南大学 | Method for high-throughput screening of eukaryotic cell and virus interaction target gene |
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| CN101985037A (en) * | 2010-05-31 | 2011-03-16 | 上海医学生命科学研究中心有限公司 | Applications of receptor for activated C-kinase 1 as target against tumor drug-resistance |
| CN106834298A (en) * | 2017-01-17 | 2017-06-13 | 西南大学 | Silkworm BmST genes and its application |
| CN112888785A (en) * | 2018-08-03 | 2021-06-01 | 联邦科学技术研究组织 | RNA molecules comprising non-canonical base pairs |
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