CN108178797B - A kind of anti-silkworm BmSRC polyclonal antiserum, preparation method and application - Google Patents

Abstract

The invention relates to an anti-silkworm BmSRC polyclonal antiserum, a preparation method and an application. The method comprises using the antigen represented by the sequence shown in SEQ ID NO: 1 to obtain the anti-silkworm BmSRC polyclonal antiserum through animal immunization. The anti-Bombyx mori BmSRC polyclonal antiserum prepared according to the above preparation method can be used to detect the conserved SRC protein in Lepidopteran insects. The method successfully clones the silkworm BmSRC gene, expresses and purifies the active protein in prokaryotic cells, and produces polyclonal antiserum by immunizing mice for many times, and can carry out experiments such as western detection of BmSRC protein expression level in vivo, and immunofluorescence of cell crawling films. All these provide useful tools for the functional study of silkworm BmSRC proteins and conserved SRC proteins in lepidopteran insects.

Description

A kind of anti-silkworm BmSRC polyclonal antiserum, preparation method and application

technical field

The present invention relates to polyclonal antiserum, and more particularly, to an anti-silkworm BmSRC polyclonal antiserum, preparation method and application.

Background technique

Scavenger receptor (SR) is a transmembrane glycoprotein located on the cell surface. It belongs to the receptor supergene family and is a multifunctional receptor. The receptor recognizes a wide range of ligands, including different gene products that bind modified LDL and specific polyanionic ligands. It mainly recognizes polyanions, including natural and modified low-density lipoproteins, apoptotic cells and pathogens. It is involved in a series of physiological and pathological processes in the body, including atherosclerosis formation or protection against host immune damage, cell adhesion, apoptotic cell clearance, tissue protection, and cell proliferation. In 1979, Goldstein et al. first discovered that there is a binding site for the uptake and degradation of acetylated low-density lipoprotein on the macrophages of mouse atherosclerotic plaques, which was later called macrophage scavenger receptor 1, which was the earliest. The discovered scavenger receptor. Subsequently, other scavenger receptors were discovered one after another, and they together formed the scavenger receptor family. According to the different domains, scavenger receptors are divided into 10 types: scavenger receptor A-J (scavenger receptor, A-J). According to the difference of amino acid structure, SR is divided into two subtypes, and the two types are slightly different: type I contains a cysteine region, while type II does not contain a cysteine region. The basic functions of the two types of SR are the same. Class C scavenger receptors (SRCs) are type I transmembrane glycoproteins comprising several distinct domains and are located on the outer surface of the cytoplasmic membrane with their C-terminal region in the cytoplasm. SRCs have been identified in several invertebrates, such as Drosophila melanogaster and Aedes aegypti, whereas SRCs have not been identified in mammals.

SUMMARY OF THE INVENTION

In view of the above problems, the present invention aims to provide an anti-Bombyx mori BmSRC polyclonal antiserum, a preparation method and an application.

To achieve the above object, the present invention adopts the following technical solutions:

On the one hand, an embodiment of the present invention discloses a method for preparing an anti-silkworm BmSRC polyclonal antiserum, which comprises obtaining the anti-silkworm BmSRC polyclonal antiserum through animal immunization using an antigen represented by the sequence shown in SEQ ID NO: 1 .

Further, the antigen is prepared by the following steps:

(1) Extracting silkworm total RNA and reverse transcription to obtain cDNA;

(2) According to the predicted target gene sequence and EST sequence shown in SEQ ID NO: 3 provided by SilkDB, design amplification primers shown in SEQ ID NO: 7-10 to obtain the first amplification product;

After the first amplification product is connected with the first connection vector, the first connection product is obtained, the first competent cell is transformed, the first positive clone is obtained, the first positive clone is sequenced and verified, and the BmSRC full-length coding gene obtained by sequencing The sequence is shown in SEQ ID NO: 6;

(3) Prokaryotic expression and protein purification of silkworm BmSRC

Select the BmSRC specific fragment to obtain the sequence shown in SEQ ID NO: 1, and design primers with Hindlll and xhoI restriction sites to carry out PCR amplification to obtain the second amplification product;

After the second amplification product is connected with the second connection vector, a second connection product is obtained, and the second connection product is transformed into a second competent cell to induce protein expression;

Purify the induced expression protein to obtain the antigen.

Further, the primer sequences with Hindll1 and xhoI restriction sites are the sequences shown in SEQ ID NOs: 11-16.

Further, the first linking vector is PMD19-T Simple vector, and the first competent cell is Trans1-T1Phage Resistant competent cell.

Further, the second connection vector is pET32a prokaryotic expression vector, the second competent cell is Rosetta strain competent, and the first connection product is PET32a-SRC.

Further, the inducible expression of the protein includes disrupting the induced cells, and performing SDS-PAGE electrophoresis on the collected supernatant and the precipitate, wherein the separation gel concentration and the shrinkage gel concentration are 10% and 5%, respectively.

Further, in the prokaryotic expression and protein purification of Bombyx mori BmSRC, the PCR amplification reaction conditions were: pre-denaturation at 94°C for 2 minutes, then denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, cycle 25. Second, the final extension at 72°C for 10 minutes, the obtained PCR product was identified and recovered by agarose gel electrophoresis.

Further, the method for extracting the total RNA of silkworm is: take the fifth instar three-day larvae of silkworm to obtain tissues and organs, and from the fourth instar three days to the upper cluster 2 days blood, the tissue and organs after the blood and liquid nitrogen grinding are added respectively. Trizol, and RNA was extracted separately.

The present invention also provides the anti-silkworm BmSRC polyclonal antiserum prepared by the above-mentioned preparation method.

The anti-Bombyx mori BmSRC polyclonal antiserum prepared according to the above preparation method can be used to detect the conserved SRC protein in Lepidopteran insects.

The beneficial effects of the present invention are:

The method successfully clones the silkworm BmSRC gene, expresses and purifies the active protein in prokaryotic cells, and produces polyclonal antiserum by immunizing mice for many times, and can carry out experiments such as western detection of BmSRC protein expression level in vivo, and immunofluorescence of cell crawling films. All these provide useful tools for the functional study of silkworm BmSRC proteins and conserved SRC proteins in lepidopteran insects.

Description of drawings

Figures 1A-1D are full-length clones of the silkworm class C scavenger receptor gene BmSRC;

Among them, Figure 1A: large fragment PCR product of BmSRC, Figure 1B: 5'RACE product of BmSRC, Figure 1C: 3'RACE product of BmSRC, Figure 1D: full length of BmSRC.

Fig. 2A-Fig. 2F are the construction and protein purification of silkworm BmSRC prokaryotic expression vector;

Among them, Figure 2A is the construction of T-BmSRC vector, M: Marker; 1: T-Simple empty plasmid; 2: T-BmSRC plasmid; 3: T-BmSRC was verified by double digestion with HindIII and XhoI; 4: BmSRC digestion recovery fragment;

Figure 2B shows the construction of the PET32a-BmSRC prokaryotic expression vector, M: Marker; 1: PET32a empty plasmid; 2: PET32a-BmSRC plasmid; 3: PET32a-BmSRC was verified by double digestion with HindIII and XhoI;

Figure 2C shows the induced expression of BmSRC in E. coli with IPTG at a final concentration of 1 mmol·L-1 at different temperatures, M: Marker; 1: Uninduced supernatant protein; 2: Uninduced inclusion body protein; 3: 1 mmol·L Supernatant protein induced by L-1IPTG at 16°C; 4: Inclusion body protein induced by 1mmol·L-1IPTG at 16°C; 5: Supernatant protein induced by 1mmol·L-1IPTG at 25°C; 6: Induction by 1mmol·L-1IPTG at 25°C 7: Supernatant protein induced by 1mmol·L-1IPTG at 37℃; 8: Inclusion body protein induced by 1mmol·L-1IPTG at 37℃;

Figure 2D shows the induced expression of BmSRC in E. coli at 37°C with different IPTG concentrations, M: Marker; 1: supernatant protein induced by 0mmol·L-1IPTG at 37°C; 2: inclusion bodies induced by 0mmol·L-1IPTG at 37°C protein; 3: supernatant protein induced by 0.1mmol·L-1IPTG at 37℃; 4: inclusion body protein induced by 0.1mmol·L-1IPTG at 37℃; 5: supernatant protein induced by 0.2mmol·L-1IPTG at 37℃; 6: Inclusion body protein induced by 0.2mmol·L-1IPTG at 37°C; 7: Supernatant protein induced by 0.4mmol·L-1IPTG at 37°C; 8: Inclusion body protein induced by 0.4mmol·L-1IPTG at 37°C; 9: 0.6mmol·L-1IPTG-induced supernatant protein at 37℃; 10: 0.6mmol·L-1IPTG-induced inclusion body protein at 37℃; 11: 0.8mmol·L-1IPTG-induced supernatant protein at 37℃; 12: 0.8mmol ·Inclusion body protein induced by L-1IPTG at 37°C; 13: Supernatant protein induced by 1mmol·L-1IPTG at 37°C; 14: Inclusion body protein induced by 1mmol·L-1IPTG at 37°C;

Figure 2E is the purification of BmSRC recombinant protein, M: Marker; 1: supernatant protein without induction; 2. inclusion body protein without induction; 3: supernatant protein induced by 0.1 mmol·L-1IPTG on a large scale; 4: 0.1mmol·L-1IPTG large-scale induced inclusion body protein;

Figure 2F shows the BmSRC protein obtained after purification. M: Marker; 5: Purified inclusion body protein.

Figure 3A-Figure 3B is the detection of anti-silkworm BmSRC protein serum;

Among them, Figure 3A is the Western blot detection of BmSRC protein expression in each tissue at 5 days and 3 days. Mi: midgut; Ha: blood; Ge: gonad; Fa: fat body; Ma: Malpighian duct; Ep: epidermis; He: head;

Figure 3B shows the expression of BmSRC in blood detected by immunofluorescence, Gr: granulosa cells; Oe: pseudoplasma cells; Pl: plasma cells.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

Example 1 Extraction of total RNA and synthesis of cDNA

The third instar larvae of the silkworm were dissected to obtain the head, epidermis, midgut, silk gland, fat body, Malpighian duct, testis, ovary, blood and other tissues and organs, as well as blood from the fourth instar three days to the upper cluster two days. In addition to adding Trizol directly, other tissues were ground with liquid nitrogen and then added with Trizol. Blood and other tissues were added to Trizol in two separate operations to extract RNA. Total RNA was extracted according to Invitrogen's TotalRNA operation guide, and the reverse transcription kit was used according to the instructions. M-MLV reverse transcriptase system was reverse transcribed to obtain cDNA.

Example 2 Amplification of the 5' end sequence and 3' end sequence of the silkworm scavenger receptor BmSRC by RACE method

According to the predicted target gene sequence and EST sequence shown in SEQ NO: 2 provided by the silkworm genome database SilkDB (http://www.silkdb.org/silkdb/), the corresponding amino acid sequence is shown in SEQ ID NO: 1, Primers were designed with Primer 5.0 software according to primer design principles. Amplify part of the intermediate sequence of the gene. After the sequence was verified to be correct, gene-specific primers GSP1, NGSP1, GSP2, and NGSP2 were designed according to this sequence. The primer sequences of the genes are shown in Table 1, SEQ NOs: 7-10. 5' and 3' RACE amplification was then performed according to the RACE kit. The amplified product was detected by 1% agarose gel electrophoresis, recovered and connected to the PMD19-T Simple vector, transformed, screened for positive clones and sent to BGI for sequencing, and the cDNA sequence was obtained after analysis and splicing, such as SEQ NO: 6, 5 The sequencing result of the 'end sequence is as SEQ NO: 4, and the sequencing result of the 3' end sequence is SEQ NO: 5. The results are shown in Figures 1A-1D. The corresponding gene number in the silkworm genome database is BGIBMGA004577-TA, located on chromosome 27, its scaffold is nscaf2800, and its position is from 531837 to 541893. The full-length cDNA sequence of this gene is 2047bp, and contains a complete open 1821bp long. The reading frame (ORF) encodes a total of 606 amino acid residues, the lengths of the 5' and 3' untranslated regions (UTR) are 399 bp and 693 bp, respectively, and the 3'-UTR structural region contains a tailed signal sequence AATAAA .

Table 1 Primer sequences

Example 3 Construction of prokaryotic expression vector and induced expression

Design corresponding primers with HindIII and XhoI restriction sites for PCR amplification. The primer sequences are shown in Table 1, as shown in SEQ NOs 11-16. PCR amplification was carried out using the blood cDNA of silkworm fourth instars as a template. The PCR amplification reaction conditions were: 94°C pre-denaturation for 2 minutes, then 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, and 72°C extension for 1 minute, a total of 25 cycle, and the final extension at 72°C for 10 minutes; the PCR product was identified and recovered by agarose gel electrophoresis, the recovered product was double digested with HindIII and XhoI, and the BmSRC gene fragment was recovered, as shown in Figure 2A. The PCR product was cloned into the prokaryotic expression vector pET32a and named pET32a-SRC, as shown in Figure 2B. The positive cloned plasmids were picked and transformed into competent Rosetta strains. A single colony was picked and inoculated in LB medium containing ampicillin for 12 hours, and then transferred to 100 μL in two 10 mL LB medium respectively. When cultured to an OD value of 0.6-1.0, IPTG with a final concentration of 1 mmol·L-1 and chloramphenicol with a final concentration of 2 μL·mL-1 were added at 300 r/min at 16 °C, 25 °C and 37 °C, respectively. nourish. Among them, the 16°C group was cultured for 20-24 hours, the 25°C group was cultured for 12-16 hours, and the 37°C group was cultured for 4-6 hours. The bacterial solution after induction was centrifuged at 5000 r/min and 4 °C for 10 min, the bacterial cells were collected, the supernatant was discarded, and washed with PBS. After resuspending the bacterial cells in 1 mL of PBS, place them on ice for sonication until the bacterial liquid becomes transparent. Centrifuge at 5000 r/min for 10 min at 4°C. The supernatant and pellet were collected separately for SDS-PAGE electrophoresis. Separating and stacking gel concentrations were 10% and 5%, respectively. Coomassie brilliant blue staining, destaining, observation results, the results are shown in Figure 2C. Subsequently, a single colony was also picked and inoculated into LB medium containing ampicillin at 37°C, shaken at 280 r/min until the OD value was 0.6-1.0, and IPTG was added to the final concentration of 0.2, 0.4, 0.6 mmol·L- 1. Induction for 6h. The induced bacterial solution was subjected to the same ultrasonic disruption, SDS-PAGE electrophoresis and Coomassie brilliant blue staining, and the results were shown in Figure 2D.

Example 4 Protein purification

Large-scale induction and expression of Bombyx mori C-type scavenger receptor recombinant protein was performed according to the condition of 0.2 mM IPTG induced at 37 °C for 6 h. Figure 2E shows the detection of the protein during large-scale induction, and it was found that the induction was successful. Then weigh the bacterial cells, mix 18 g of the bacterial cells with 250 mL of 50 mM Tris-HCL (without adjusting the pH, a little higher pH is better) buffer (the ratio with the bacterial cells is 1:10-20), and dissolve after repeated freezing and thawing in liquid nitrogen three times. , use a homogenizer to mix it, shake it in a shaker at 37 °C for 1 h, take it out and add DNase (10 μg/mL), during the homogenization process, add 250 μL 1M MgCl2 (final concentration 1mM), slowly add After lysozyme (0.1 mg/ml), adjust the pH to about 9.5-10. Put it into a 37°C shaker for about 1 hour, and stir until it is not sticky; take it out and pre-cool at -20°C for 20 minutes, sonicate the bacteria to pure clearness, freeze and centrifuge at high speed, and collect the precipitates separately. The protein inclusion bodies were washed with 5% acetic acid and 1% Triron at a mass-to-volume ratio of 1:20 for 30 min, followed by high-speed freezing and centrifugation, and the procedure was repeated once after collecting the precipitate. The pellet was then washed once with 2M urea (containing 25mM Tris, 5mM EDTA), refrigerated and centrifuged at high speed to collect the pellet. The pellet was collected by washing with 20 mM Tris, 0.5 M NaCl and 50% ethanol, pH 7.0, high-speed refrigerated centrifugation. The precipitate was washed with double distilled water to remove salt, and the precipitate was collected by high-speed refrigerated centrifugation. Dissolve the inclusion bodies with 8M urea (containing 25Mm Tris-HCl, 0.3M NaCl, pH 8.0), adjust the pH to 11.5-12.0, stir overnight, freeze and centrifuge (15000rpm, 30min) the next day, and use 0.22μm micropores for the supernatant. After filtration through the filter membrane, it is ready for use. Finally, the recombinant protein was purified with Ni ⁺ column (Ni-NTA) (according to the operating instructions provided by GE), the eluate was collected, and the components of the eluted protein were analyzed by SDS-PAGE. Figure 2F shows the inclusion bodies obtained after purification. protein.

Example 5 Antiserum preparation

The purified recombinant protein was used to inject 3 healthy adult male Kunming mice. Before the injection, the tail tip of each mouse was cut to take blood, and about 50 μL of normal serum was collected as a negative control. After standing at room temperature, the supernatant was collected by centrifugation. Mixed with glycerol at a volume ratio of 1:1 and stored at –80°C.

When the mice were immunized, each mouse was intraperitoneally injected with protein 5 times, and the cycles were 0, 10, 10, 15 and 21 days. The first time was mixed with equal volume of Freund's complete adjuvant, and the last four times were mixed with equal volume of Freund's incomplete adjuvant. One week after the last immunization, the eyeballs were enucleated for blood collection, left standing at room temperature for 2 h, and then centrifuged at 3 000 r/min for 10 min to remove the serum, add 30% glycerol, and store at -80°C.

Example 6 Detection of anti-Bombyx mori BmSRC protein serum

The protein of each tissue (including midgut, blood cells, gonads, fat body, Malpighian duct, epidermis, and head) of silkworm 5th instar 3 days was used as samples, and the anti-BmSRC protein serum of silkworm was detected by Western blot, and the protein was electrophoresed by SDS-PAGE and transferred. After membrane, block with TBST (Nacl8.8g, 1M Tris-HCl (pH8.0) 20ml, 0.5ml Tween20, dilute to 1L) containing 5% BSA at room temperature for 2h, and the antiserum produced by the mouse was 1:1: After 5000 dilution, incubate at 4°C overnight, wash 3 times with TBST for 10 minutes each time, incubate with HRP-labeled mouse IgG diluted at a ratio of 1:10000 for 1 hour at room temperature, and wash again with TBST for 3 times for 10 minutes each time, Using ECL chromogenic solution for color development and exposure with an imager, as shown in Figure 3A, the antiserum produced by the mouse can specifically display the BmSRC protein band in each tissue of silkworm at 5 instar and 3 days, with a size of about 60kD.

488 Donkey Anti-Mouse IgG(H+L)抗体室温孵育1小时，用PBS洗3次，每次10分钟；用PBS以1：2000比例的稀释Hoechst33342室温孵育40分钟后，PBS洗3次，每次10分钟，添加抗荧光淬灭剂然后用指甲油封边后用荧光显微镜成像观察，如图3B所示，BmSRC抗血清可以特异的标记家蚕血液中的BmSRC蛋白，并且标记颗粒细胞和拟浆细胞，并显示其表达于家蚕血液细胞的细胞膜上。We also performed immunofluorescence detection on the antiserum of silkworm BmSRC protein. The blood cells of silkworm 4 instars were used as samples. After the cells adhered, the slides were washed three times with PBS. After each 5 minutes, they were incubated with PBS containing 4% paraformaldehyde for 15 minutes at room temperature, and then washed with PBS for 5 minutes. After 3 times, the mice were blocked with PBS containing 1% BSA and 10% goat serum for 2 hours at room temperature, and the antiserum produced by the mouse was diluted 1:750 with the blocking solution. 3 washes for 10 minutes each time with Alexa

488 Donkey Anti-Mouse IgG (H+L) antibody was incubated at room temperature for 1 hour, washed three times with PBS for 10 minutes each time; Hoechst33342 diluted with PBS at a ratio of 1:2000 was incubated at room temperature for 40 minutes, washed with PBS three times, each time For 10 minutes, add anti-fluorescence quencher, then seal the edges with nail polish and observe with a fluorescence microscope. As shown in Figure 3B, BmSRC antiserum can specifically label BmSRC protein in silkworm blood, as well as granulosa cells and plasma-like cells. , and showed that it was expressed on the cell membrane of silkworm blood cells.

To sum up, the present invention successfully clones the silkworm BmSRC gene, expresses and purifies the active protein in prokaryotic cells, and produces polyclonal antiserum by immunizing mice multiple times. Fluorescence and other experiments. All these provide useful tools for the functional study of silkworm BmSRC proteins and conserved SRC proteins in lepidopteran insects.

The above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention; if the present invention is modified or equivalently replaced without departing from the spirit and scope of the present invention, it shall be covered by the claims of the present invention. within the scope of protection.

sequence listing

<110> Southwest University

<120> A kind of anti-silkworm BmSRC polyclonal antiserum, preparation method and application

<130> 17P99761-CN

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 532

<212> PRT

<213> Artificial Sequence

<400> 1

Phe Ala Leu Arg Cys Pro Tyr Pro Tyr Leu Gln His Gly Lys Ala Arg

1 5 10 15

Leu Arg Thr Lys Ser Arg Ile Val Lys Phe Val Cys Asn Pro Arg Tyr

20 25 30

Lys Leu Val Gly Asn Lys Tyr Ser Ile Cys Arg Met Gly Arg Trp Glu

35 40 45

Glu Gln Leu Pro Val Cys Val Lys Ser Gly Cys Pro Lys Leu Pro Pro

50 55 60

Ile Gln Met Thr His His Asp Gly Ala Trp Leu Met Thr Phe Cys Leu

65 70 75 80

Pro Asn Tyr Arg Leu Glu Gly Ser Glu Val Leu Tyr Cys Asn Gly Tyr

85 90 95

Arg Trp Asn Ser Thr Ala Pro Lys Cys Val Glu Met Asn Asn Asn Val

100 105 110

Thr Thr Ile Lys Tyr Ser Cys Asp Phe Glu Glu Asp Leu Cys Gly Trp

115 120 125

Ile Gln Asp Glu Phe His Asp Phe Asp Trp Lys Arg Leu Asn Thr Lys

130 135 140

Thr Pro Ser Ser Phe Thr Leu Thr Gly Pro Trp Phe Asp His Thr Tyr

145 150 155 160

Gly Ser Arg Gly Lys Gly His Phe Met Tyr Ile Glu Ser Thr Gly Arg

165 170 175

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

180 185 190

Ile Ala Lys Asp Gly Cys Phe Gln Leu Tyr Tyr His Met Tyr Gly Arg

195 200 205

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

210 215 220

Met Thr Met Leu Ala Ser Glu Glu Gln Arg Lys Asp Tyr Ile Ile Phe

225 230 235 240

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

245 250 255

Leu Lys Asp Phe Asn Glu Asp Phe Gln Ile Val Ile Glu Gly Ile Arg

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Ile Thr Arg Gly Cys Gly Cys Asp Gly Asp Cys Val Ile Tyr Gly Tyr

325 330 335

Cys Cys Gln Asp Phe Val Asp Leu Cys Ile Glu Lys Asp Pro Glu Thr

340 345 350

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

355 360 365

Lys Leu Val Ala Ser Thr Thr Asn Ala Thr Thr Ser Thr Ser Thr Thr

370 375 380

Thr Thr Thr Thr Pro Lys Pro Ile Val Ile Leu Ser Thr Lys Thr Thr

385 390 395 400

Thr Thr Thr Ala Ser Pro Lys Pro Thr Thr Pro Thr Thr Thr Ser Thr

405 410 415

Lys Thr Phe Pro Thr Ile Pro Thr Arg Thr Thr Thr Ile Thr Val Ser

420 425 430

Lys Thr Pro Lys Thr Ser Thr Lys Thr Thr Ile Ala Arg Arg Ser Ser

435 440 445

Ser Pro Ser Gln Lys Gln Thr Thr Lys Val Leu Lys Pro Thr Thr Gln

450 455 460

Ser Ser Ser Ile Thr Pro Lys Ser Ala Thr Arg Lys Val Thr Ser Val

465 470 475 480

Ala Ser Thr Thr Val Gly Ser Lys Lys Leu Thr Ser Ser Thr Thr Val

485 490 495

Lys Pro Ala Ser Thr Ser Lys Lys Val Thr Glu Lys Gln Ser Phe Thr

500 505 510

Lys Lys Met Thr Val Asp Val Phe Val Ala Lys Lys Ser Lys Gly Ala

515 520 525

Ser Lys Thr Leu

530

<210> 2

<211> 1596

<212> DNA

<213> Artificial Sequence

<400> 2

ttcgctctaa gatgtcctta cccatatcta cagcatggca aagctagatt gcgaactaag 60

tccagaatcg tgaagttcgt atgtaaccct agatataaat tggttgggaa caaatactct 120

atatgtcgaa tggggcgttg ggaggagcag ctgccggtct gtgttaaatc aggctgccct 180

aaattgccac caattcagat gacgcatcac gatggcgctt ggctcatgac gttctgcctc 240

ccgaactaca gattagaggg ttcagaagtg ttatactgca acggatacag atggaatagc 300

actgcgccta aatgcgttga gatgaacaac aacgtaacaa cgatcaagta cagttgcgac 360

ttcgaagagg acctctgcgg ttggatacag gacgagttcc acgatttcga ttggaagcga 420

ctgaacacga aaactccgag ctcctttacg cttaccggac cgtggtttga tcacacgtac 480

ggctccagag gaaagggcca tttcatgtat atcgagagta ctgggcgttt cattaacgat 540

acagcccgac ttttatcgcc gatttacgat tcggccatag cgaaggacgg atgttttcag 600

ctttattatc acatgtatgg aagatcatta ggaggcctta gggtatacca aaaaccggac 660

agcgtcgatt taatgacgat gctggcttca gaggaacagc gaaaggatta cattattttt 720

gaaaagtggg gcgaacaagg tgacctctgg ctttccgctg ctccacgact aaaggacttt 780

aacgaagact ttcagatagt gatcgaagga atccgaggat ccagtttcat gagcgacctg 840

gctattgacg atatttccgt gttgagagga cagaagtgcg tagacgcagc ggcgcatgca 900

atcaccccct ctccttataa agccgactct tgcgtcggtc gatgcttcca aaatataact 960

atcacaagag gctgtggttg cgacggagac tgcgtcatct atggctactg ctgtcaagac 1020

ttcgtagatc tatgcatcga aaaagatccc gaaacaacag tgattgccga aactggttcg 1080

accgttgcat taccgcaaac acagaaacta gtagcttcta caaccaacgc tacaacttca 1140

acttcaacca caacgacgac tacacccaaa cccattgtta tactttcaac taaaactacc 1200

actactactg cttctcccaa acctacgact cccactacca ccagtactaa gacttttcct 1260

actattccca caaggaccac tactatcaca gtcagtaaaa ctccaaaaac cagtaccaaa 1320

acgaccatag ccagacgttc ctctagccca tcccagaaac agaccaccaa ggtccttaag 1380

cctacgactc agtccagttc catcacacct aagagtgcaa ctcgtaaggt gacgtctgtc 1440

gcttcgacca cagttgggag taaaaaatta acatccagca ctacagtaaa acctgctagt 1500

accagcaaaa aggttactga aaaacagtct tttaccaaga agatgacggt ggacgttttt 1560

gttgcgaaga aatcgaaagg agcctcaaaa acactg 1596

<210> 3

<211> 1563

<212> DNA

<213> Artificial Sequence

<400> 3

atgacgcatc acgatggcgc ttggctcatg acgttctgcc tcccgaacta cagattagag 60

ggttcagaag tgttatactg caacggatac agatggaata gcactgcgcc taaatgcgtt 120

gagatgaaca acaacgtaac aacgatcaag tacagttgcg acttcgaaga ggacctctgc 180

ggttggatac aggacgagtt ccacgatttc gattggaagc gactgaacac gaaaactccg 240

agctccttta cgcttaccgg accgtggttt gatcacacgt acggctccag aggaaagggc 300

catttcatgt atatcgagag tactgggcgt ttcattaacg atacagcccg acttttatcg 360

ccgatttacg attcggccat agcgaaggac ggatgttttc agctttatta tcacatgtat 420

ggaagatcat taggaggcct tagggtatac caaaaaccgg acagcgtcga tttaatgacg 480

atgctggctt cagaggaaca gcgaaaggat tacattattt ttgaaaagtg gggcgaacaa 540

ggtgacctct ggctttccgc tgctccacga ctaaaggact ttaacgaaga ctttcagata 600

gtgatcgaag gaatccgagg atccagtttc atgagcgacc tggctattga cgatatttcc 660

gtgttgagag gacagaagtg cgtagacgca gcggcgcatg caatcacccc ctctccttat 720

aaagccgact cttgcgtcgg tcgatgcttc caaaatataa ctatcacaag aggctgtggt 780

tgcgacggag actgcgtcat ctatggctac tgctgtcaag acttcgtaga tctatgcatc 840

gaaaaagatc ccgaaacaac agtgattgcc gaaactggtt cgaccgttgc attaccgcaa 900

acacagaaac tagtagcttc tacaaccaac gctacaactt caacttcaac cacaacgacg 960

actacaccca aacccattgt tatactttca actaaaacta ccactactac tgcttctccc 1020

aaacctacga ctcccactac caccagtact aagacttttc ctactattcc cacaaggacc 1080

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 tgatt 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.

Images