Preparation method of squaliobarbus curriculus IPS-1 gene polyclonal antibody

文档序号：1485253 发布日期：2020-02-28 浏览：14次中文

阅读说明：本技术 赤眼鳟ips-1基因多克隆抗体的制备方法 (Preparation method of squaliobarbus curriculus IPS-1 gene polyclonal antibody ) 是由肖调义赵鑫李耀国于 2019-11-16 设计创作，主要内容包括：本发明公开了赤眼鳟IPS-1基因多克隆抗体的制备方法,步骤如下：(1)赤眼鳟IPS-1基因的克隆；(2)赤眼鳟IPS-1基因重组表达载体的构建；(3)赤眼鳟IPS-1蛋白表达及破菌流程；(4)重组蛋白的纯化及浓度测定；(5)多克隆抗体的制备。本发明成功制备了赤眼鳟IPS-1基因多克隆抗体,进一步探究赤眼鳟干扰素启动刺激因子1应对GCRV等病原物的免疫作用,为鱼类抗病育种提供抗性分子资源。(The invention discloses a preparation method of a squaliobarbus curriculus IPS-1 gene polyclonal antibody, which comprises the following steps: (1) cloning Oncorhynchus mykiss IPS-1 gene; (2) constructing a recombinant expression vector of the Oncorhynchus mykiss IPS-1 gene; (3) carrying out IPS-1 protein expression and bacterium breaking process on the squaliobarbus curriculus; (4) purifying and measuring the concentration of the recombinant protein; (5) and (4) preparing a polyclonal antibody. The invention successfully prepares the polyclonal antibody of the squaliobarbus curriculus IPS-1 gene, further explores the immune function of squaliobarbus curriculus interferon promoter-stimulating factor 1 on pathogens such as GCRV and the like, and provides resistance molecular resources for fish breeding for disease resistance.)

1. The preparation method of the squaliobarbus curriculus IPS-1 gene polyclonal antibody is characterized by comprising the following steps:

(1) cloning of Oncorhynchus mykiss IPS-1 Gene

Extracting total RNA of spleen tissues of the squaliobarbus curriculus, performing reverse transcription to synthesize a first chain of 5 'and 3' cDNA, and designing two pairs of specific primers: IPS-1-5 '(W) and IPS-1-3' (W), IPS1-1-5 '(N) and IPS 1-1-3' (N), the nucleotide sequences are respectively shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4; performing first 5 'RACE-PCR and 3' RACE-PCR amplification by using IPS-1-5 '(W) and IPS-1-3' (W) to obtain products as templates of second PCR; performing a second round of 5 'RACE-PCR and 3' RACE-PCR amplification by using a second pair of specific primers IPS1-1-5 '(N) and IPS 1-1-3' (N), purifying PCR products, connecting T vectors, transforming and sequencing; splicing to obtain the full-length cDNA sequence of the squaliobarbus curriculus IPS-1 gene;

(2) construction of squaliobarbus curriculus IPS-1 gene recombinant expression vector

Designing a third pair OF specific primers IPS-1-OF and IPS-1-OR, wherein the nucleotide sequences are respectively shown as SEQ ID No.5 and SEQ ID No.6, respectively adding BamH I and Xho I enzyme cutting sites to the upstream primer and the downstream primer, carrying out PCR amplification by taking spleen tissue cDNA OF the squaliobarbus curriculus obtained in the step (1) as a template and IPS-1-OF and IPS-1-OR as primers, carrying out double enzyme cutting on a PCR product and a pET-28a (+) expression vector, recovering and purifying, connecting T4 ligase, transforming escherichia coli DH5 α, and obtaining a recombinant expression vector pET-28 a-SUMO-IPS-1;

(3) oncorhynchus mykiss IPS-1 protein expression and bacterium breaking process

Transforming the recombinant expression vector pET-28a-SUMO-IPS-1 obtained in the step (2) into escherichia coli BL21(DE3), culturing in LB culture solution containing 50mg/L kanamycin until OD value is 0.6, and adding IPTG for induction; centrifugally collecting thalli; determining the protein expression position and purity by SDS-PAGE electrophoresis after bacteria breaking;

(4) purification and concentration determination of recombinant proteins

Purifying the processing methods of loading, eluting, processing and regenerating the recombinant protein by using a purification kit; adopting Lowry method to determine the protein concentration; setting standard protein solutions with different concentrations, drawing a standard curve, and calculating the protein concentration of the sample according to the light absorption value of the sample and the standard curve;

(5) preparation of polyclonal antibodies

And (3) taking the squaliobarbus curriculus pET-28a-SUMO-IPS-1 recombinant protein purified in the step (4) as an antigen, immunizing a New Zealand rabbit, separating whole blood, collecting rabbit antiserum, and purifying to obtain the squaliobarbus curriculus IPS1 polyclonal antibody.

2. The method of claim 1, wherein in step (1), the first round of 5 'RACE-PCR and 3' RACE-PCR amplification reaction is performed at 94 ℃ for 5 min; 5 cycles of 94 ℃ for 30s and 72 ℃ for 3 min; 5 cycles of 94 ℃ for 30s, 70 ℃ for 30s, and 72 ℃ for 3 min; 30 cycles of 94 ℃ for 30s, 68 ℃ for 30s, and 72 ℃ for 3 min.

3. The method according to claim 1, wherein in step (1), the second round of 5 'RACE-PCR and 3' RACE-PCR amplification reaction conditions are 94 ℃ for 5 min; 30 cycles of 94 ℃ 30s, 68 ℃ 30s, 72 ℃ 3min, then 72 ℃ 7 min.

4. The preparation method according to claim 1, wherein in the step (3), the bacteria breaking method comprises the following steps: 50mL of 1 XPBS suspension bacterial liquid is taken and 50 mu L of mercaptoethanol is added; setting the bacteria breaking time to be 3s by using an ultrasonic breaking and breaking instrument, and carrying out ultrasonic breaking for 10min at intervals of 3 s; centrifuging for 10min to obtain No.1 supernatant and precipitate, placing No.1 supernatant in a 50mL centrifuge tube, and storing at 4 deg.C temporarily; washing the precipitate twice with deionized water, weighing 6g of urea, dissolving with 1 × PBS, diluting to a constant volume of 44mL, pouring the precipitate, and blowing and suspending the precipitate; continuously breaking the bacteria for 5min according to the setting of 3s for breaking the bacteria and 3s for interval; after the bacteria are broken, centrifuging at 12000r/min for 10min to obtain No.2 supernatant and inclusion bodies, sucking out the No.2 supernatant, and temporarily storing at 4 ℃ for later use; add 6mL ddH to Inclusion bodies₂After O suspension, evenly loading the mixture into 4 centrifuge tubes, and centrifuging the mixture for 2min at 12000 r/min; discarding the supernatant, selecting one tube, adding 1.5mL of 8M urea, dissolving, centrifuging at 12000r/min for 1min, and transferring the supernatant into a new centrifuge tube.

5. The method according to claim 1, wherein in the step (5), the New Zealand white rabbit is immunized by: the purified squaliobarbus curriculus pET-28a-SUMO-IPS-1 recombinant protein is used as an antigen, and 8 times of immunization is carried out on a New Zealand white rabbit according to the volume ratio of 1: 1, adding Freund's complete adjuvant, and adopting subcutaneous multi-point injection on the back; freund incomplete adjuvant is selected for enhancing immunity; the last immunization is carried out for 10 days.

6. The primer pair for cloning the full-length cDNA sequence of the Oncorhynchus mykiss IPS-1 gene is characterized in that the primer pair has two groups, namely IPS-1-5 '(W) and IPS-1-3' (W), IPS1-1-5 '(N) and IPS 1-1-3' (N), and the nucleotide sequences of the IPS-1-5 '(W) and the IPS-1-3' (W) are respectively shown as SEQ ID No.1 and SEQ ID No. 2; the nucleotide sequences of IPS1-1-5 '(N) and IPS 1-1-3' (N) are shown as SEQ ID No.3 and SEQ ID No.4 respectively.

7. The primer pair for cloning the Oncorhynchus mykiss IPS-1 gene is characterized in that the primer pair is IPS-1-OF and IPS-1-OR, and the nucleotide sequences are respectively shown as SEQ ID No.5 and SEQ ID No. 6.

8. The use of the primer pair of claim 6 or 7 for cloning the IPS-1 gene of the squaliobarbus curriculus.

Technical Field

The invention belongs to the field of preparation of polyclonal antibodies, and particularly relates to a preparation method of a trout IPS-1 gene polyclonal antibody.

Background

Grass carp is often killed by infection with Grass Carp Reovirus (GCRV) with a double-stranded RNA genome. The squaliobarbus curriculus and the grass carp belong to the subfamily of the Atlantic subfamily, have strong resistance to GCRV, can be used as a resistance resource donor to be hybridized with the grass carp to obtain strong-resistance offspring, and are excellent materials for researching resistance molecular mechanisms.

The squaliobarbus curriculus Interferon promoter stimulator 1 (IPS 1) is used as a joint molecule of a retinoic acid-induced gene (RIG-I) like receptor (RIG-I likereceptors, RLRs) family to participate in immune response against GCRV.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to provide a method for preparing a polyclonal antibody against squaliobarbus curriculus interferon stimulating factor 1 aims to further explore the immune effect of squaliobarbus curriculus interferon stimulating factor 1 on pathogens such as GCRV and the like and provide resistance molecule resources for fish disease-resistant breeding.

The technical scheme of the invention is as follows: the preparation method of the squaliobarbus curriculus IPS-1 gene polyclonal antibody comprises the following steps:

(1) cloning of Oncorhynchus mykiss IPS-1 Gene

(2) construction of squaliobarbus curriculus IPS-1 gene recombinant expression vector

(3) oncorhynchus mykiss IPS-1 protein expression and bacterium breaking process

(4) purification and concentration determination of recombinant proteins

(5) preparation of polyclonal antibodies

Further, in the step (1), the first round of 5 'RACE-PCR and 3' RACE-PCR amplification reaction program is 94 ℃ for 5 min; 5 cycles of 94 ℃ for 30s and 72 ℃ for 3 min; 5 cycles of 94 ℃ for 30s, 70 ℃ for 30s, and 72 ℃ for 3 min; 30 cycles of 94 ℃ for 30s, 68 ℃ for 30s, and 72 ℃ for 3 min.

Further, in the step (1), the amplification reaction conditions of the second round of 5 'RACE-PCR and 3' RACE-PCR are 94 ℃ for 5 min; 30 cycles of 94 ℃ 30s, 68 ℃ 30s, 72 ℃ 3min, then 72 ℃ 7 min.

Further, in the step (3), the bacteria breaking method comprises the following steps: 50mL of 1 XPBS suspension bacterial liquid is taken and 50 mu L of mercaptoethanol is added; setting the bacteria breaking time to be 3s by using an ultrasonic breaking and breaking instrument, and carrying out ultrasonic breaking for 10min at intervals of 3 s; centrifuging for 10min to obtain No.1 supernatant and precipitate, placing No.1 supernatant in a 50mL centrifuge tube, and storing at 4 deg.C temporarily; washing the precipitate twice with deionized water, weighing 6g of urea, dissolving with 1 × PBS, diluting to a constant volume of 44mL, pouring the precipitate, and blowing and suspending the precipitate; continuously breaking the bacteria for 5min according to the setting of 3s for breaking the bacteria and 3s for interval; after the bacteria are broken, centrifuging at 12000r/min for 10min to obtain No.2 supernatantAnd inclusion body, sucking out No.2 supernatant, and temporarily storing at 4 ℃ for later use; add 6mL ddH to Inclusion bodies₂After O suspension, evenly loading the mixture into 4 centrifuge tubes, and centrifuging the mixture for 2min at 12000 r/min; discarding the supernatant, selecting one tube, adding 1.5mL of 8M urea, dissolving, centrifuging at 12000r/min for 1min, and transferring the supernatant into a new centrifuge tube.

Further, in the step (5), the method for immunizing the New Zealand white rabbits comprises the following steps: the purified squaliobarbus curriculus pET-28a-SUMO-IPS-1 recombinant protein is used as an antigen, and 8 times of immunization is carried out on a New Zealand white rabbit according to the volume ratio of 1: 1, adding Freund's complete adjuvant, and adopting subcutaneous multi-point injection on the back; freund incomplete adjuvant is selected for enhancing immunity; the last immunization is carried out for 10 days.

Two groups of primer pairs are provided for cloning the full-length cDNA sequence of the Oncorhynchus mykiss IPS-1 gene, namely IPS-1-5 '(W) and IPS-1-3' (W), IPS1-1-5 '(N) and IPS 1-1-3' (N), and the nucleotide sequences of the IPS-1-5 '(W) and the IPS-1-3' (W) are respectively shown as SEQ ID No.1 and SEQ ID No. 2; the nucleotide sequences of IPS1-1-5 '(N) and IPS 1-1-3' (N) are shown as SEQ ID No.3 and SEQ ID No.4 respectively.

The primer pair for the squaliobarbus curriculus IPS-1 gene cloning is IPS-1-OF and IPS-1-OR, and the nucleotide sequences are respectively shown as SEQ ID No.5 and SEQ ID No. 6.

The primer pair is applied to the clone of the Oncorhynchus mykiss IPS-1 gene.

Compared with the prior art, the invention has the following beneficial effects:

the invention successfully prepares the polyclonal antibody of the squaliobarbus curriculus IPS-1 gene, further explores the immune function of squaliobarbus curriculus interferon promoter-stimulating factor 1 on pathogens such as GCRV and the like, and provides resistance molecular resources for fish breeding for disease resistance.

Drawings

FIG. 1 shows the identification result after bacteria breaking and purification. 1: supernatant No. 1; 2: supernatant No. 2; 3: inclusion bodies diluted 2-fold; 4: inclusion bodies at 5-fold dilution; 5: inclusion bodies diluted 10-fold; 6: pET-28a-SUMO no-load induction expression; 7: 0.4mg/mL BSA; 8: and (5) Marker.

FIG. 2 Western blot to detect polyclonal antibody to Oncorhynchus squamosa IPS1 in tissues. C IPS-1: a healthy squaliobarbus curriculus sample; 12h IPS-1: GCRV stimulates squaliobarbus curriculus samples for 12 h.

FIG. 3 shows that Western blot detects polyclonal antibody of Oncorhynchus squamosa IPS1 in cells. pEGFP-C1-IPS-1: cell samples transfected with pEGFP-C1-IPS-1.

Detailed Description

The reagents and equipment used in the present invention are commercially available unless otherwise specified, and the method is a conventional method known to those skilled in the art. The primers used in the present invention are shown in Table 1.

Table 1 primer sequences are shown in table 1 below:

primer name	Primer sequence (5'-3')
		IPS-1-5’(W)	CTCCAAATCTTCAGAGACCACGGAGCAAGG(SEQ ID No.1)
IPS-1-3’(W)	GGTTACGCACCCTCCCTCTTCCATCGAGCA(SEQ ID No.2)
		IPS1-1-5’(N)	GGCTCCTGAACTGTCCTTTCCTCTCTCACT(SEQ ID No.3)
IPS1-1-3’(N)	ATGCTCCACACAATGCTGCTTCCTCGAAT(SEQ ID No.4)
		IPS-1-OF	TCGAGCTCAAGCTTCGAATTCATGTCATTGACACGTGAACAATTTT(SEQ ID No.5)
IPS-1-OR	TTATCTAGATCCGGTGGATCCATGCTTGAGCTTCCAAGCCA(SEQ ID No.6)

1. Cloning of Oncorhynchus mykiss IPS-1 Gene

Extracting total RNA of spleen tissue of the squaliobarbus curriculus, synthesizing a first chain of 5 'cDNA and a first chain of 3' cDNA by using a SMARTer RACE cDNA amplification kit, and storing at-80 ℃; according to the reported IPS-1 gene sequence in GenBank database, two pairs of specific primers are designed: IPS-1-5 '(W), IPS-1-3' (W), IPS1-1-5 '(N) and IPS 1-1-3' (N), the primer sequences are shown in Table 1. IPS-1-5 '(W) and IPS-1-3' (W) were subjected to a first round of 5 'RACE-PCR and 3' RACE-PCR amplification with a reaction program of 94 ℃ for 5 min; 5 cycles of 94 ℃ for 30s and 72 ℃ for 3 min; 5 cycles of 94 ℃ for 30s, 70 ℃ for 30s, and 72 ℃ for 3 min; 30 cycles of 94 ℃ for 30s, 68 ℃ for 30s, and 72 ℃ for 3 min. Obtaining a product diluted by 50 times and using the product as a template of the second round of PCR; performing a second round of 5 'RACE-PCR and 3' RACE-PCR amplification by using a second pair of specific primers IPS1-1-5 '(N) and IPS 1-1-3' (N), wherein the reaction condition is 94 ℃ for 5 min; 30 cycles of 94 ℃ 30s, 68 ℃ 30s, 72 ℃ 3min, then 72 ℃ 7 min. The PCR product was purified, ligated to T-vector, transformed and sequenced. Splicing to obtain the full-length cDNA sequence of the squaliobarbus curriculus IPS-1 gene.

2. Construction of squaliobarbus curriculus IPS-1 gene recombinant expression vector

Designing a third pair OF specific primers IPS-1-OF and IPS-1-OR according to the full-length cDNA sequence OF the IPS-1 gene OF the squaliobarbus curriculus, respectively adding BamH I and Xho I enzyme cutting sites to the upstream and downstream primers, carrying out PCR amplification by using squaliobarbus curriculus spleen tissue cDNA as a template and IPS-1-OF and IPS-1-OR as primers, carrying out double enzyme cutting on a PCR product and a pET-28a (+) expression vector, recovering and purifying, connecting T4 ligase, and transforming escherichia coli DH5 α to obtain a recombinant expression vector pET-28 a-SUMO-IPS-1.

3. Oncorhynchus mykiss IPS-1 protein expression and bacterium breaking process

Transforming the obtained recombinant expression vector pET-28a-SUMO-IPS-1 into Escherichia coli BL21(DE3), culturing in LB culture solution containing 50mg/L kanamycin until OD value is 0.6, and adding IPTG for induction; centrifugally collecting thalli;

and (3) a bacterium breaking process: 50mL of 1 XPBS suspension was added to 50. mu.L of mercaptoethanol. And (3) utilizing an ultrasonic bacteria breaking and crushing instrument, setting bacteria breaking time for 3s at an interval of 3s, and carrying out ultrasonic bacteria breaking for 10 min. Centrifuging for 10min to obtain No.1 supernatant and precipitate, placing No.1 supernatant in a 50mL centrifuge tube, and storing at 4 deg.C temporarily; the precipitate was washed twice with deionized water, then 6g of urea was weighed and dissolved in 1 × PBS to a constant volume of 44mL, poured into the precipitate, and the precipitate was suspended by blowing. Continuously breaking the bacteria for 5min according to the setting of 3s for breaking the bacteria and 3s for interval; after the bacteria are broken, centrifuging at 12000r/min for 10min to obtain No.2 supernatant and inclusion bodies, sucking out the No.2 supernatant, and temporarily storing at 4 ℃ for later use. Add 6mL ddH to Inclusion bodies₂After O suspension, the suspension is loaded into 4 2mL centrifuge tubes on average and centrifuged at 12000r/min for 2 min. Abandoning the supernatant, selecting one tube, adding 1.5mL of 8M urea into the tube, dissolving, centrifuging at 12000r/min for 1min, and transferring the supernatant into a new 2mL centrifuge tube. SDS-PAGE was used to determine the location and purity of protein expression (FIG. 1).

4. Purification and concentration determination of recombinant proteins

Purifying the processing methods of loading, eluting, processing and regenerating the recombinant protein by using a purification kit; protein concentration was measured by Lowry method. Setting standard protein solutions with different concentrations, drawing a standard curve, and calculating the protein concentration of the sample according to the light absorption value of the sample and the standard curve.

5. Preparation of polyclonal antibodies

The purified squaliobarbus curriculus pET-28a-SUMO-IPS-1 recombinant protein is used as an antigen, and 8 times of immunization is carried out on a New Zealand white rabbit according to the volume ratio of 1: 1, adding Freund's complete adjuvant, and adopting subcutaneous multi-point injection on the back; freund incomplete adjuvant is selected for enhancing immunity; and after 10 days of the last immunization, separating whole blood, collecting rabbit antiserum, and purifying to obtain the squaliobarbus curriculus IPS1 polyclonal antibody.

6. Western blot detection

Total proteins of liver, spleen and head kidney tissues of the healthy squaliobarbus curriculus after being stimulated by GCRV for 12h are respectively extracted, and the protein concentration is determined by using a Lowry method. The protein sample was mixed with a 5 × loading buffer at a ratio of 4:1 and denatured by boiling water bath for 5 minutes. Taking 20 mu L of the denatured sample, and carrying out SDS-PAGE electrophoresis on 8% separating gel; stopping electrophoresis for membrane transfer when bromophenol blue runs to the bottom of the separation gel; and sealing the membrane after the membrane is rotated. Then, incubating the Oncorhynchus mykiss IPS-1 polyclonal antibody for 2h at room temperature; then, incubating rabbit secondary antibody for 1h at room temperature; finally, detection was performed using ECL luminescence reagent (fig. 2). Meanwhile, a squaliobarbus curriculus IPS1 overexpression vector pEGFP-C1-ScIPS-1 is constructed, and Lipofectamine 2000 is used for transfection in grass carp ovarian cells. After 24h of transfection, total cell protein was extracted and detected using the Oncorhynchus squaliosus IPS-1 polyclonal antibody as described above (FIG. 3).

The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.

Sequence listing

<110> Hunan agriculture university

Preparation method of <120> squaliobarbus curriculus IPS-1 gene polyclonal antibody

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