CN108178797B - Polyclonal antiserum for resisting bombyx mori BmSRC, preparation method and application - Google Patents

Abstract

The invention relates to a polyclonal antiserum for resisting bombyx mori BmSRC, a preparation method and application, wherein the method comprises the following steps of adopting SEQ ID NO:1, and obtaining the polyclonal antiserum for resisting the bombyx mori BmSRC by animal immunization. The polyclonal antiserum for resisting silkworm BmSRC prepared by the preparation method can be used for detecting conserved SRC protein in lepidoptera insects. The invention successfully clones the Bombyx mori BmSRC gene, carries out prokaryotic expression and purification to obtain active protein, generates polyclonal antiserum by immunizing a mouse for multiple times, and can carry out experiments such as western detection of the expression level of the BmSRC protein in vivo, immunofluorescence of a cell slide and the like. These all provide an advantageous tool for functional studies of bombyx mori bmssrc proteins and SRC proteins conserved in lepidopteran insects.

Description

Polyclonal antiserum for resisting bombyx mori BmSRC, preparation method and application

Technical Field

The invention relates to polyclonal antiserum, and more particularly relates to polyclonal antiserum for resisting bombyx mori BmSRC, a preparation method and application.

Background

The Scavenger Receptor (SR) is a transmembrane glycoprotein located on the cell surface, belongs to the receptor supergene family, and is a multifunctional receptor. The ligands recognized by this receptor are very broad and include different gene products that bind modified LDL and specific polyanionic ligands. Mainly to recognize polyanions, including natural and modified low density lipoproteins, apoptotic cells, pathogens, and the like. It is involved in a range of physiological and pathological processes in the body including atherogenesis or protection of host immune damage defense, cell adhesion, apoptotic cell clearance, tissue protection and cell proliferation. Goldstein et al first discovered in 1979 that binding sites for the uptake and degradation of acetylated low density lipoproteins exist on macrophages in atherosclerotic plaques in mice, and were later referred to as macrophage scavenger receptor 1, the earliest scavenger receptor discovered. Subsequently, other scavenger receptors were discovered, which together form a scavenger receptor family. Depending on the domain, scavenger receptors are divided into 10 types: scavenger receptors a-J (a-J). The SR is divided into two subtypes according to the difference of amino acid structures, the two subtypes are slightly different, the type I contains a cysteine region, the type II does not contain the cysteine region, and the two subtypes have the same SR basic function. Class C Scavenger Receptors (SRC) are type i transmembrane glycoproteins that contain several distinct domains and are located on the outer surface of the cytoplasmic membrane with the C-terminal region in the cytoplasm. SRC has been identified in several invertebrates, such as Drosophila melanogaster and Aedes aegypti, however SRC has not been found in mammals.

Disclosure of Invention

The invention aims to solve the problems and aims to provide an anti-bombyx mori BmSRC polyclonal antiserum, a preparation method and application.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the embodiments of the present invention disclose a method for preparing anti-bombyx mori bmssr polyclonal antiserum, which comprises the steps of using SEQ ID NO:1, and obtaining the polyclonal antiserum for resisting the bombyx mori BmSRC by animal immunization.

Further, the antigen is prepared by the following steps:

(1) extracting total RNA of silkworm and reverse transcribing to obtain cDNA;

(2) according to SEQ ID NO: 3, predicting a target gene sequence and an EST sequence, designing an amplification primer shown as SEQ ID NO:7-10, and obtaining a first amplification product;

connecting the first amplification product with a first connecting vector to obtain a first connecting product, transforming a first competent cell to obtain a first positive clone, carrying out sequencing verification on the first positive clone, wherein the sequence of the full-length coding gene of the BmSRC obtained by sequencing is shown as SEQ ID NO 6;

(3) prokaryotic expression and protein purification of bombyx mori BmSRC

Selecting a BmSRC specific fragment to obtain a sequence shown as SEQ ID NO. 1, and designing a primer with Hindlll and xhoI enzyme cutting sites for PCR amplification to obtain a second amplification product;

connecting the second amplification product with a second connecting vector to obtain a second connecting product, and transforming a second competent cell by the second connecting product to perform protein induction expression;

and purifying the protein subjected to induced expression to obtain the antigen.

Further, the primer sequences with Hindlll and XhoI cleavage sites are shown in SEQ ID Nos. 11-16.

Further, the first connection carrier is a PMD19-T Simple carrier, and the first competent cell is a Trans1-T1Phage resist competent cell.

Further, the second ligation vector is a pET32a prokaryotic expression vector, the second competent cell is competent by a Rosetta strain, and the first ligation product is PET32 a-SRC.

Further, the protein induction expression comprises the step of carrying out SDS-PAGE electrophoresis on respectively collected supernatant and precipitate after breaking the induced thallus, wherein the concentration of separation gel and the concentration of contraction gel are respectively 10% and 5%.

Further, in the prokaryotic expression and protein purification of bombyx mori BmSRC, the PCR amplification reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min, denaturation at 94 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1 min, circulation for 25 times, and final extension at 72 ℃ for 10min, and the obtained PCR product is identified and recovered by agarose gel electrophoresis.

Further, the method for extracting the total RNA of the silkworms comprises the following steps: and (3) obtaining tissue organs of five-instar three-day larvae of the silkworms and blood from four-instar three days to 2 days of upper cluster, adding Trizol to the blood and the tissue organs ground by liquid nitrogen respectively, and extracting RNA respectively.

The invention also provides the polyclonal antiserum for resisting the bombyx mori BmSRC, which is prepared by the preparation method.

The polyclonal antiserum for resisting silkworm BmSRC prepared by the preparation method can be used for detecting conserved SRC protein in lepidoptera insects.

The invention has the beneficial effects that:

the invention successfully clones the Bombyx mori BmSRC gene, carries out prokaryotic expression and purification to obtain active protein, generates polyclonal antiserum by immunizing a mouse for multiple times, and can carry out experiments such as western detection of the expression level of the BmSRC protein in vivo, immunofluorescence of a cell slide and the like. These all provide an advantageous tool for functional studies of bombyx mori bmssrc proteins and SRC proteins conserved in lepidopteran insects.

Drawings

FIGS. 1A to 1D are full-length clones of the Bombyx mori class C scavenger receptor gene BmSRC;

wherein, fig. 1A: large fragment PCR product of BmSRC, fig. 1B: 5' RACE product of BmSRC, FIG. 1C: 3' RACE product of BmSRC, fig. 1D: full length of BmSRC.

FIGS. 2A-2F show construction of Bombyx mori BmSRC prokaryotic expression vector and protein purification;

wherein, FIG. 2A is the T-BmSRC vector construction, M: marker; 1: T-Simple empty plasmid; 2: a T-BmSRC plasmid; 3: the T-BmSRC is verified by double enzyme digestion of HindIII and XhoI; 4: carrying out BmSRC enzyme digestion to recover fragments;

FIG. 2B is a PET32a-BmSRC prokaryotic expression vector construct, M: marker; 1: PET32a empty plasmid; 2: PET32a-BmSRC plasmid; 3: PET32a-BmSRC was verified by double digestion with HindIII and XhoI;

FIG. 2C is the induced expression of BmSRC at different temperatures in E.coli with IPTG at a final concentration of 1 mmol.L-1, M: marker; 1: (ii) uninduced supernatant protein; 2: (ii) uninduced inclusion body protein; 3: 1 mmol. L-1IPTG16 deg.C induced supernatant protein; 4: 1mmol L-1IPTG16 deg.C induced inclusion body protein; 5: 1 mmol. L-1IPTG 25 ℃ induced supernatant protein; 6: 1mmol L-1IPTG 25 deg.C induced inclusion body protein; 7: 1 mmol. L-1IPTG 37 ℃ induced supernatant protein; 8: 1 mmol. L-1IPTG inclusion body protein induced at 37 deg.c;

FIG. 2D is the induced expression of BmSRC at different IPTG concentrations in E.coli at 37 deg.C, M: marker; 1: 0 mmol. L-1IPTG 37 deg.C induced supernatant protein; 2: 0mmol of inclusion body protein induced by L-1IPTG at 37 ℃; 3: 0.1mmol of L-1IPTG supernatant protein induced at 37 ℃; 4: 0.1mmol of inclusion body protein induced by L-1IPTG at 37 ℃; 5: 0.2mmol of L-1IPTG induced supernatant protein at 37 ℃; 6: 0.2mmol of inclusion body protein induced by L-1IPTG at 37 ℃; 7: 0.4mmol of L-1IPTG induced supernatant protein at 37 ℃; 8: 0.4mmol of inclusion body protein induced by L-1IPTG at 37 ℃; 9: 0.6mmol of L-1IPTG supernatant protein induced at 37 ℃; 10: 0.6mmol of inclusion body protein induced by L-1IPTG at 37 ℃; 11: 0.8mmol of L-1IPTG 37 ℃ induced supernatant protein; 12: 0.8mmol of inclusion body protein induced by L-1IPTG at 37 ℃; 13: 1 mmol. L-1IPTG 37 ℃ induced supernatant protein; 14: 1 mmol. L-1IPTG inclusion body protein induced at 37 deg.c;

fig. 2E shows BmSRC recombinant protein purification, M: marker; 1: (ii) an uninduced supernatant protein; 2. inclusion body proteins that are not induced; 3: 0.1 mmol.L-1 IPTG large-scale induced supernatant protein; 4: inclusion body protein induced by 0.1 mmol.L-1 IPTG in large scale;

FIG. 2F is the purified BmSRC protein. M: marker; 5: the resulting inclusion body protein was purified.

FIGS. 3A-3B are assays for anti-bombyx mori BmSRC protein serum;

wherein, FIG. 3A shows the expression of the BmSRC protein in each tissue at 5-day-old for 3 days by Western blot detection. Mi: the middle intestine; ha: blood; ge: the gonad; fa: a fat body; and Ma: a Martensitic tube; ep: a epidermis; he: a head;

FIG. 3B shows the expression of BmSRC in blood detected by immunofluorescence, Gr: granulosa cells; and Oe: a plasmacytoid cell; pl: plasma cells.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

EXAMPLE 1 extraction of Total RNA and cDNA Synthesis

Dissecting five-year-three-day larva of silkworm to obtain tissue organs such as head, epidermis, midgut, silk gland, fat body, Marek's tube, testis, ovary and blood from four-year-three-day to 2-day upper cluster, adding Trizol to the blood and other tissues after grinding with liquid nitrogen, adding Trizol to the blood and other tissues twice, extracting RNA, extracting total RNA according to the TotalRNA operating manual of Invitrogen company, and performing reverse transcription by using M-MLV reverse transcriptase system according to the instruction of a reverse transcription kit to obtain cDNA.

Example 2 RACE amplification of 5 'and 3' terminal sequences of the silkworm scavenger receptor BmSRC

According to a predicted target gene sequence and an EST sequence which are provided by a silkworm genome database SilkDB (http:// www.silkdb.org/Silkbb /) and are shown as SEQ NO. 2, and a corresponding amino acid sequence is shown as SEQ ID NO. 1, a Primer is designed by using Primer 5.0 software according to a Primer design principle. Amplifying to obtain partial intermediate sequence of the gene. After the sequencing verification is correct, gene specific primers GSP1, NGSP1, GSP2 and NGSP2 are designed according to the sequences, the primer sequences of the genes are shown in table 1, and SEQ NO is 7-10. 5 'and 3' RACE amplifications were then performed according to the RACE kit. The amplified product is detected by 1% agarose gel electrophoresis, recovered and connected to a PMD19-T Simple vector, transformed, screened for positive clone and sent to Huada gene for sequencing, and after analysis and splicing, a cDNA sequence is obtained, such as SEQ NO 6, and the sequencing result of 5 'terminal sequence is shown as SEQ NO 4, and the sequencing result of 3' terminal sequence is shown as SEQ NO 5. The results are shown in FIGS. 1A to 1D. The corresponding gene number in the silkworm genome database is BGIBMGA004577-TA, which is positioned on chromosome 27, the scaffold is nscaf2800, the position is from 531837 to 541893, the cDNA full-length sequence of the gene is 2047bp, contains 1 complete Open Reading Frame (ORF) with the length of 1821bp, totally encodes 606 amino acid residues, the lengths of the 5 ' untranslated region sequence (UTR) and the 3 ' untranslated region sequence (UTR) are 399bp and 693bp respectively, and the 3 ' -UTR structural region contains 1 tailing signal sequence AATAAA.

TABLE 1 primer sequences

Example 3 prokaryotic expression vector construction and inducible expression

Primers with HindIII and XhoI restriction sites were designed for PCR amplification, and the primer sequences are shown in Table 1 and shown in SEQ NO 11-16. Carrying out PCR amplification by using the four-age dormancy blood cDNA of the silkworm as a template, wherein the PCR amplification reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min, then denaturation at 94 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1 min for 25 cycles, and finally extension at 72 ℃ for 10 min; the PCR product was identified by agarose gel electrophoresis and recovered, the recovered product was double digested with HindIII and XhoI, and the BmSRC gene fragment was recovered as shown in FIG. 2A. The PCR product was cloned into the prokaryotic expression vector pET32a, designated pET32a-SRC, as shown in FIG. 2B. Positive cloning plasmid is picked, Rosetta strain is transformed to be competent, and a single colony is picked and inoculated in LB culture medium containing ampicillin for 12h, and then is respectively transferred into two 10mL LB culture media by 100 mu L. When the culture was carried out until the OD value was 0.6-1.0, IPTG having a final concentration of 1 mmol. multidot.L-1 and chloramphenicol having a final concentration of 2. multidot.L. multidot.mL-1 were added, and the culture was carried out at 16 ℃ at 25 ℃ and 37 ℃ at 300r/min, respectively. Wherein, the group culture at 16 ℃ is 20-24h, the group culture at 25 ℃ is 12-16h, and the group culture at 37 ℃ is 4-6 h. And centrifuging the induced bacterial liquid at 5000r/min and 4 ℃ for 10min, collecting thalli, discarding supernatant, and washing with PBS. After resuspending the cells with 1ml PBS, the cells were sonicated on ice until the cells became clear. Centrifuging at 5000r/min at 4 deg.C for 10 min. The supernatant and the precipitate were collected separately and subjected to SDS-PAGE. The concentrations of the separation gel and the concentration gel were 10% and 5%, respectively. Coomassie brilliant blue staining, decoloration and observation result, and the result is shown in figure 2C. Subsequently, a single colony was also picked and inoculated in LB medium containing ampicillin at 37 ℃ and cultured with shaking at 280r/min until OD value was 0.6-1.0, IPTG was added to final concentrations of 0.2, 0.4 and 0.6 mmol.L-1, respectively, and induction was carried out for 6 hours. The induced suspension was subjected to the same ultrasonication, SDS-PAGE and Coomassie blue staining, and the results are shown in FIG. 2D.

EXAMPLE 4 protein purification

The large-scale induction expression of silkworm C-class scavenger receptor recombinant protein was carried out according to the condition of 0.2mM IPTG induction at 37 ℃ for 6h, and FIG. 2E shows the detection of the protein during the large-scale induction, and the induction was found to be successful. Then, the cells were weighed, 18g of the cells were mixed with 250mL of 50mM Tris-HCl buffer (without pH adjustment, with a slightly higher pH) (in a ratio of 1:10-20) and frozen and thawed three times with liquid nitrogen, and then dissolved, and the mixture was mixed by a homogenizer, and shaken at 37 ℃ for 1 hour in a shaker, and then taken out and added with DNase (10. mu.g/mL), 250. mu.L of 1M MgCl2 (final concentration: 1mM) was added during the homogenization, lysozyme (0.1mg/mL) was slowly added, and then the pH was adjusted to about 9.5-10. Reacting in a shaking table at 37 ℃ for about 1h, and stirring until the mixture is not sticky; precooling at-20 deg.C for 20min, ultrasonically crushing bacteria to pure clear, high-speed freezing and centrifuging, and collecting precipitate respectively. The mixture was diluted with a mixture containing 5% acetic acid, 1% trinon at 1: washing the protein inclusion body for 30min at the mass-to-volume ratio of 20, freezing and centrifuging at a high speed, collecting the precipitate, and repeating the step once. The pellet was then washed once with 2M urea (25 mM Tris, 5mM EDTA included), and the pellet was collected by high speed refrigerated centrifugation. The pellet was collected by washing with 20mM Tris, 0.5M NaCl and 50% ethanol, pH 7.0, high speed refrigerated centrifugation. Washing the precipitate with double distilled water to remove salt, high speed freezing and centrifuging, and collecting the precipitate. The inclusion bodies were solubilized with 8M urea (25 mM Tris-HCl, 0.3M NaCl, pH8.0), pH adjusted to 11.5-12.0, stirred overnight, frozen and centrifuged the next day (15000rpm, 30min), and the supernatant was filtered through a 0.22 μ M microfiltration membrane for use. Finally using Ni⁺The recombinant protein was purified by column (Ni-NTA) (according to the instructions provided by GE company), the eluate was collected, and the fractions of the eluted protein were analyzed by SDS-PAGE, and FIG. 2F shows the inclusion body protein obtained after purification.

Example 5 antiserum preparation

Injecting 3 healthy adult male Kunming mice with the purified recombinant protein, taking blood from the tip of the tail before injection of each mouse, collecting about 50 μ L of normal serum as a negative control, standing at room temperature, centrifuging to collect supernatant, mixing the supernatant with glycerol at a volume ratio of 1:1, and storing at-80 ℃.

Mice were immunized with 5 injections of protein per abdominal cavity for periods of 0, 10, 15 and 21 d. The 1 st time is mixed with the Freund complete adjuvant in equal volume, and the last 4 times are mixed with the Freund incomplete adjuvant in equal volume. After the last 1-time immunization for 1 week, removing the eyeball, taking blood, standing at room temperature for 2h, centrifuging at 3000 r/min for 10min, taking out the serum, adding 30% glycerol, and subpackaging at-80 deg.C for preservation.

Example 6 detection of anti-Bombyx mori BmSRC protein serum

Western blot detection is carried out on silkworm BmSRC protein serum by using silkworm tissue (comprising midgut, blood cells, gonad, fat body, Marsdenia tube, epidermis and head) protein of 3 days after 5 days of age of silkworm as a sample, the protein is subjected to SDS-PAGE electrophoresis and membrane transfer, blocking with TBST (Nacl8.8g,1M Tris-HCl (pH8.0)20ml, 0.5ml Tween20, constant volume to 1L) containing 5% BSA at room temperature for 2h, diluting mouse-produced antiserum at 1:5000, incubating overnight at 4 ℃, washing 3 times with TBST, each for 10 minutes, after incubation of HRP-labeled mouse IgG diluted at a ratio of 1:10000 for 1 hour at room temperature, washing with TBST for 3 times, each for 10min, developing with ECL developing solution, exposing with imager, as shown in FIG. 3A, antisera raised in mice specifically showed a BmSRC protein band of approximately 60kD in 5-year-old, 3-day silkworm tissues.

We also performed immunofluorescence detection on bombyx mori bmscr protein antiserum. Using blood cells of 4-year-old bombyx mori as a sample, washing the cells with PBS 3 times after the cells are attached to the wall, incubating the cells with PBS containing 4% paraformaldehyde for 15 minutes at room temperature after 5 minutes each time, washing the cells with PBS for 5 minutes, washing the cells for 3 times, blocking the cells with PBS blocking solution containing 1% BSA and 10% goat serum for 2 hours at room temperature, and blocking the cells with the blocking solution at a rate of 1: antiserum raised from mice was diluted 750, incubated overnight at 4 ℃, washed 3 times with PBS for 10 minutes each, and then Alexa

The 488 Donkey Anti-Mouse IgG (H + L) antibody was incubated at room temperature for 1 hour, washed 3 times with PBS, each for 10 minutes; with PBS at 1: after incubation at room temperature for 40 minutes at 2000 scale dilution Hoechst33342, 3 washes with PBS for 10 minutes each, addition of an anti-fluorescence quencher followed by edge sealing with nail polish and visualization by fluorescence microscopy, as shown in FIG. 3B, the BmSRC antiserum can be specifically labeledThe BmSRC protein in the blood of the silkworm is recorded, granular cells and plasma-like cells are marked, and the expression of the BmSRC protein on the cell membrane of the blood cells of the silkworm is shown.

In conclusion, the invention successfully clones the bombyx mori BmSRC gene, carries out prokaryotic expression and purification to obtain active protein, generates polyclonal antiserum by immunizing a mouse for multiple times, and can carry out experiments such as western detection of the expression level of the BmSRC protein in vivo, immunofluorescence of a cell slide and the like. These all provide an advantageous tool for functional studies of bombyx mori bmssrc proteins and SRC proteins conserved in lepidopteran insects.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; it is intended that the following claims be interpreted as including all such alterations, modifications, and equivalents as fall within the true spirit and scope of the invention.

Sequence listing

<110> university of southwest

<120> polyclonal antiserum for resisting bombyx mori BmSRC, and preparation method and application thereof

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Phe Ile Asn Asp Thr Ala Arg Leu Leu Ser Pro Ile Tyr Asp Ser Ala

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Ser Leu Gly Gly Leu Arg Val Tyr Gln Lys Pro Asp Ser Val Asp Leu

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Glu Lys Trp Gly Glu Gln Gly Asp Leu Trp Leu Ser Ala Ala Pro Arg

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Gly Ser Ser Phe Met Ser Asp Leu Ala Ile Asp Asp Ile Ser Val Leu

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Arg Gly Gln Lys Cys Val Asp Ala Ala Ala His Ala Ile Thr Pro Ser

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Pro Tyr Lys Ala Asp Ser Cys Val Gly Arg Cys Phe Gln Asn Ile Thr

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Thr Val Ile Ala Glu Thr Gly Ser Thr Val Ala Leu Pro Gln Thr Gln

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ggtgacctct ggctttccgc tgctccacga ctaaaggact ttaacgaaga ctttcagata 600

gtgatcgaag gaatccgagg atccagtttc atgagcgacc tggctattga cgatatttcc 660

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actactatca cagtcagtaa aactccaaaa accagtacca aaacgaccat agccagacgt 1140

tcctctagcc catcccagaa acagaccacc aaggtcctta agcctacgac tcagtccagt 1200

tccatcacac ctaagagtgc aactcgtaag gtgacgtctg tcgcttcgac cacagttggg 1260

agtaaaaaat taacatccag cactacagta aaacctgcta gtaccagcaa aaaggttact 1320

gaaaaacagt cttttaccaa gaagatgacg gtggacgttt ttgttgcgaa gaaatcgaaa 1380

ggagcctcaa aaacactgca aacgtctggc ataatagttg gtgtgttgtt ttgtgtattt 1440

gcgttggtcg gatccgtggt ggcttggcga cgttacggcg gcatgatgat cgttagaagg 1500

ttgagaggtc aaatcgcgaa tgaccctgaa gtgcgctatc tgagtgccca tatggacgat 1560

tga 1563

<210> 4

<211> 399

<212> DNA

<213> Artificial Sequence

<400> 4

ggacactgac atggactgaa ggagtagaaa atttttgaaa atgttctgac accgcgaata 60

gttaagtcta aattcggaaa tgacatgaac ggattaaatt atataaaaaa ataattttga 120

acaaaacttt aaaaaaaaga agtgaaacat gaatttatta tataagtgta ttttatcgtt 180

aatgtttttt tgttatatcg aggcccaatt cgctctaaga tgtccttacc catatctaca 240

gcatggcaaa gctagattgc gaactaagtc cagaatcgtg aagttcgtat gtaaccctag 300

atataaattg gttgggaaca aatactctat atgtcgaatg gggcgttggg aggagcagct 360

gccggtctgt gttaaatcag gctgccctaa attgccacc 399

<210> 5

<211> 700

<212> DNA

<213> Artificial Sequence

<400> 5

accacaacga cgactacacc caaacccatt gttatacttt caactaaaac taccactact 60

actgcttctc ccaaacctac gactcccact accaccagta ctaagacttt tcctactatt 120

cccacaagga ccactactat cacagtcagt aaaactccaa aaaccagtac caaaacgacc 180

atagccagac gttcctctag cccatcccag aaacagacca ccaaggtcct taagcctacg 240

actcagtcca gttccatcac acctaagagt gcaactcgta aggtgacgtc tgtcgcttcg 300

accacagttg ggagtaaaaa attaacatcc agcactacag taaaacctgc tagtaccagc 360

aaaaaggtta ctgaaaaaca gtcttttacc aagaagatga cggtggacgt ttttgttgcg 420

aagaaatcga aaggagcctc aaaaacactg caaacgtctg gcataatagt tggtgtgttg 480

ttttgtgtat ttgcgttggt cggatccgtg gtggcttggc gacgttacgg cggcatgatg 540

atcgttagaa ggttgagagg tcaaatcgcg aatgaccctg aagtgcgcta tctgagtgcc 600

catatggacg attgaactgt tcttttaaaa taaactattt tagctttaaa aaaaaggaag 660

aaaaaaatag taataaccac tgtcatgccg ttacgtagcg 700

<210> 6

<211> 2047

<212> DNA

<213> Artificial Sequence

<400> 6

ggacactgac atggactgaa ggagtagaaa atttttgaaa atgttctgac accgcgaata 60

gttaagtcta aattcggaaa tgacatgaac ggattaaatt atataaaaaa ataattttga 120

acaaaacttt aaaaaaaaga agtgaaacat gaatttatta tataagtgta ttttatcgtt 180

aatgtttttt tgttatatcg aggcccaatt cgctctaaga tgtccttacc catatctaca 240

gcatggcaaa gctagattgc gaactaagtc cagaatcgtg aagttcgtat gtaaccctag 300

atataaattg gttgggaaca aatactctat atgtcgaatg gggcgttggg aggagcagct 360

gccggtctgt gttaaatcag gctgccctaa attgccacca attcagatga cgcatcacga 420

tggcgcttgg ctcatgacgt tctgcctccc gaactacaga ttagagggtt cagaagtgtt 480

atactgcaac ggatacagat ggaatagcac tgcgcctaaa tgcgttgaga tgaacaacaa 540

cgtaacaacg atcaagtaca gttgcgactt cgaagaggac ctctgcggtt ggatacagga 600

cgagttccac gatttcgatt ggaagcgact gaacacgaaa actccgagct cctttacgct 660

taccggaccg tggtttgatc acacgtacgg ctccagagga aagggccatt tcatgtatat 720

cgagagtact gggcgtttca ttaacgatac agcccgactt ttatcgccga tttacgattc 780

ggccatagcg aaggacggat gttttcagct ttattatcac atgtatggaa gatcattagg 840

aggccttagg gtataccaaa aaccggacag cgtcgattta atgacgatgc tggcttcaga 900

ggaacagcga aaggattaca ttatttttga aaagtggggc gaacaaggtg acctctggct 960

ttccgctgct ccacgactaa aggactttaa cgaagacttt cagatagtga tcgaaggaat 1020

ccgaggatcc agtttcatga gcgacctggc tattgacgat atttccgtgt tgagaggaca 1080

gaagtgcgta gacgcagcgg cgcatgcaat caccccctct ccttataaag ccgactcttg 1140

cgtcggtcga tgcttccaaa atataactat cacaagaggc tgtggttgcg acggagactg 1200

cgtcatctat ggctactgct gtcaagactt cgtagatcta tgcatcgaaa aagatcccga 1260

aacaacagtg attgccgaaa ctggttcgac cgttgcatta ccgcaaacac agaaactagt 1320

agcttctaca accaacgcta caacttcaac ttcaaccaca acgacgacta cacccaaacc 1380

cattgttata ctttcaacta aaactaccac tactactgct tctcccaaac ctacgactcc 1440

cactaccacc agtactaaga cttttcctac tattcccaca aggaccacta ctatcacagt 1500

cagtaaaact ccaaaaacca gtaccaaaac gaccatagcc agacgttcct ctagcccatc 1560

ccagaaacag accaccaagg tccttaagcc tacgactcag tccagttcca tcacacctaa 1620

gagtgcaact cgtaaggtga cgtctgtcgc ttcgaccaca gttgggagta aaaaattaac 1680

atccagcact acagtaaaac ctgctagtac cagcaaaaag gttactgaaa aacagtcttt 1740

taccaagaag atgacggtgg acgtttttgt tgcgaagaaa tcgaaaggag cctcaaaaac 1800

actgcaaacg tctggcataa tagttggtgt gttgttttgt gtatttgcgt tggtcggatc 1860

cgtggtggct tggcgacgtt acggcggcat gatgatcgtt agaaggttga gaggtcaaat 1920

cgcgaatgac cctgaagtgc gctatctgag tgcccatatg gacgattgaa ctgttctttt 1980

aaaataaact attttagcta gaaaaaaaaa aaaaaaaaaa aaaacactgt catgccgtta 2040

cgtagcg 2047

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 7

ggagccgtac gtgtgatcaa acca 24

<210> 8

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 8

ggtggcaatt tagggcagcc tgattt 26

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 9

cgactcttgc gtcggtcgat gctt 24

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 10

accacaacga cgactacacc caaa 24

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 11

accgtgccag gctgtacta 19

<210> 12

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 12

atttctgcgg gcgtttt 17

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 13

atgaatttat tatataagtg tattt 25

<210> 14

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 14

atcgtccata tgggcac 17

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 15

ttcgctctaa gatgtcctta cc 22

<210> 16

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 16

cagtgttttt gaggctcc 18

Claims (10)

1. A preparation method of polyclonal antiserum for resisting silkworm BmSRC is characterized by comprising the following steps of: 1, and obtaining the anti-bombyx mori BmSRC polyclonal antiserum through animal immunization.

2. The method of claim 1, wherein the antigen is produced by:

(1) extracting total RNA of silkworm and reverse transcribing to obtain cDNA;

(2) according to SEQ ID NO: 3, predicting a target gene sequence and an EST sequence, designing an amplification primer shown as SEQ ID NO:7-10, and obtaining a first amplification product;

connecting the first amplification product with a first connecting vector to obtain a first connecting product, transforming a first competent cell to obtain a first positive clone, carrying out sequencing verification on the first positive clone, wherein the sequence of the full-length coding gene of the BmSRC obtained by sequencing is shown as SEQ ID NO 6;

(3) prokaryotic expression and protein purification of bombyx mori BmSRC

Selecting a BmSRC specific fragment to obtain a sequence shown as SEQ ID NO. 1, and designing a primer with Hindlll and xhoI enzyme cutting sites for PCR amplification to obtain a second amplification product;

connecting the second amplification product with a second connecting vector to obtain a second connecting product, and transforming a second competent cell by the second connecting product to perform protein induction expression;

and purifying the protein subjected to induced expression to obtain the antigen.

3. The method of claim 2, wherein the primer sequence with Hindlll and XhoI cleavage sites is as shown in SEQ ID NO. 11-16.

4. The method of claim 2, wherein the first ligation vector is a PMD19-T Simple vector and the first competent cell is a Trans1-T1Phage resist competent cell.

5. The method of claim 2, wherein the second ligation vector is a pET32a prokaryotic expression vector and the second competent cell is competent for the Rosetta strain.

6. The method of claim 2, wherein the inducing expression of the protein comprises disrupting the induced bacteria and performing SDS-PAGE on the supernatant and the pellet, wherein the gel concentration is 10% and the gel concentration is 5% respectively.

7. The method of claim 2, wherein in the prokaryotic expression and protein purification of bombyx mori bmssrc, the PCR amplification reaction conditions are: pre-denaturation at 94 ℃ for 2 min, denaturation at 94 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1 min, circulation for 25 times, and final extension at 72 ℃ for 10min, and the obtained PCR product is identified and recovered by agarose gel electrophoresis.

8. The method of claim 2, wherein the method for extracting total RNA of silkworms comprises: and (3) obtaining tissue organs of five-instar three-day larvae of the silkworms and blood from four-instar three days to 2 days of upper cluster, adding Trizol to the blood and the tissue organs ground by liquid nitrogen respectively, and extracting RNA respectively.

9. Polyclonal antiserum against bombyx mori bmstrc, prepared by the preparation process according to any one of claims 2 to 8.

10. Use of polyclonal antiserum raised against bombyx mori bmssrc prepared by the preparation method according to any one of claims 2 to 8 for the detection of bmssrc proteins in bombyx mori.

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