阅读说明：本技术 一种莱茵衣藻ift144蛋白多克隆抗体及其制备方法和应用 (Chlamydomonas reinhardtii IFT144 protein polyclonal antibody and preparation method and application thereof ) 是由 冯雳 丁梅 胡长峰 邢俊俏 李丽丽 花梦婷 陈龙 蒋俊龙 石畅 于 2019-09-30 设计创作，主要内容包括：本发明公开了一种莱茵衣藻IFT144蛋白多克隆抗体及其制备方法和应用,属于生物工程领域。该莱茵衣藻IFT144蛋白多克隆抗体的氨基酸序列如序列表中SEQ ID NO：1所示。该方法包括：构建重组表达载体,重组表达载体的核苷酸序列包括如序列表中SEQ ID NO：2所示的序列；将重组表达载体转化至感受态细胞中,得到重组表达菌株；诱导重组表达菌株的蛋白表达,得到莱茵衣藻IFT144蛋白；将莱茵衣藻IFT144蛋白作为抗原,并进行免疫处理,得到莱茵衣藻IFT144蛋白多克隆抗体。该莱茵衣藻IFT144蛋白多克隆抗体能特异性识别莱茵衣藻IFT144蛋白,且效价高和特异性好；该制备方法通过PCR扩增克隆得到编码莱茵衣藻IFT144蛋白的cDNA序列,再通过构建重组表达载体,从而通过体外表达和纯化获得了制备抗体的抗原。(The invention discloses a chlamydomonas reinhardtii IFT144 protein polyclonal antibody, a preparation method and application thereof, and belongs to the field of bioengineering. The amino acid sequence of the chlamydomonas reinhardtii IFT144 protein polyclonal antibody is shown as SEQ ID NO:1 is shown. The method comprises the following steps: constructing a recombinant expression vector, wherein the nucleotide sequence of the recombinant expression vector comprises the nucleotide sequence shown as SEQ ID NO: 2; transforming the recombinant expression vector into a competent cell to obtain a recombinant expression strain; inducing the protein expression of the recombinant expression strain to obtain Chlamydomonas reinhardtii IFT144 protein; and (3) taking the Chlamydomonas reinhardtii IFT144 protein as an antigen, and performing immune treatment to obtain the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody. The Chlamydomonas reinhardtii IFT144 protein polyclonal antibody can specifically recognize Chlamydomonas reinhardtii IFT144 protein, and has high titer and good specificity; the preparation method obtains a cDNA sequence for coding the Chlamydomonas reinhardtii IFT144 protein through PCR amplification cloning, and obtains the antigen for preparing the antibody through in vitro expression and purification by constructing a recombinant expression vector.)

1. The Chlamydomonas reinhardtii IFT144 protein polyclonal antibody is characterized in that the amino acid sequence of the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody is shown as SEQ ID NO 1 in a sequence table.

2. A method of producing a polyclonal antibody to chlamydomonas reinhardtii IFT144 protein according to claim 1, said method comprising:

constructing a recombinant expression vector, wherein the nucleotide sequence of the recombinant expression vector comprises a sequence shown as SEQ ID NO. 2 in a sequence table;

transforming the recombinant expression vector into a competent cell to obtain a recombinant expression strain;

inducing the protein expression of the recombinant expression strain to obtain Chlamydomonas reinhardtii IFT144 protein;

and taking the Chlamydomonas reinhardtii IFT144 protein as an antigen, and performing immune treatment to obtain the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody.

3. The polyclonal antibody against Chlamydomonas reinhardtii IFT144 protein of claim 2, wherein the method for constructing the recombinant expression vector comprises: extracting total RNA of chlamydomonas reinhardtii;

performing reverse transcription by taking the total RNA of the chlamydomonas reinhardtii as a template to obtain chlamydomonas reinhardtii cDNA;

performing PCR amplification by using the Chlamydomonas reinhardtii cDNA as a template to obtain a Chlamydomonas reinhardtii IFT144 target gene;

and connecting the chlamydomonas reinhardtii IFT144 target gene with a prokaryotic expression vector to obtain a recombinant expression vector.

4. The Chlamydomonas reinhardtii IFT144 protein polyclonal antibody of claim 3, wherein the prokaryotic expression vector is a double-restriction enzyme digested Escherichia coli expression vector pET-28 a.

5. The Chlamydomonas reinhardtii IFT144 protein polyclonal antibody of claim 3, wherein an upstream primer and a downstream primer are adopted for amplification during PCR amplification, the sequence of the upstream primer is shown as SEQ ID NO. 3 in the sequence table, and the sequence of the downstream primer is shown as SEQ ID NO. 4 in the sequence table.

6. The polyclonal antibody against Chlamydomonas reinhardtii IFT144 protein of claim 3, wherein the method for inducing protein expression of the recombinant expression strain comprises: culturing the recombinant expression strain at 37 ℃ overnight, then transferring the recombinant expression strain into a small shake flask of an LB liquid culture medium containing 50 mu g/mL kanamycin to prepare a bacterial liquid, and when the concentration of the bacterial liquid reaches OD₆₀₀And when the concentration is 0.6-0.8, adding isopropyl- β -D-thiogalactoside with the final concentration of 0.1-1 mM for induction, wherein the induction temperature is 37 ℃, and the induction time is 3h, so as to obtain the Chlamydomonas reinhardtii IFT144 protein.

7. The Chlamydomonas reinhardtii IFT144 protein polyclonal antibody of claim 2, wherein the method further comprises: purifying the obtained chlamydomonas reinhardtii IFT144 protein; the purification method comprises the following steps: performing first centrifugation on the obtained chlamydomonas reinhardtii IFT144 protein to obtain first centrifugal precipitate;

carrying out second centrifugation after the first centrifugal precipitation is re-suspended and washed by PBS (phosphate buffer solution), so as to obtain second centrifugal precipitation;

re-suspending the second centrifugal precipitate by using the PBS, and adding lysozyme to obtain a re-suspension;

adding PMSF into the heavy suspension, performing ultrasonic treatment, adding Triton X-100 and DTT, and centrifuging to obtain a precipitate;

performing denaturation and dissolution on the precipitate by using a denaturation solution to obtain a denaturation solution;

diluting and renaturing the denatured solution by using renaturation solution to obtain renaturation solution;

and carrying out column chromatography separation on the renaturation solution, and eluting to obtain the purified Chlamydomonas reinhardtii IFT144 protein.

8. The Chlamydomonas reinhardtii IFT144 protein polyclonal antibody of claim 2, wherein the method of immunization comprises: mixing the chlamydomonas reinhardtii IFT144 protein antigen with complete Freund's adjuvant to obtain a first mixed solution, injecting the first mixed solution into an immune animal body, mixing the IFT144 protein antigen with the incomplete Freund's adjuvant after 12 days to obtain a second mixed solution, injecting the second mixed solution into the immune animal body, mixing the IFT144 protein antigen with the incomplete Freund's adjuvant after 14 days to obtain a third mixed solution, injecting the third mixed solution into the immune animal body, mixing the IFT144 protein antigen with the incomplete Freund's adjuvant after 14 days to obtain a fourth mixed solution, and injecting the fourth mixed solution into the immune animal body.

9. The Chlamydomonas reinhardtii IFT144 protein polyclonal antibody of claim 8, wherein the immunized animal is a New Zealand white rabbit.

10. Use of a polyclonal antibody to the chlamydomonas reinhardtii IFT144 protein of claim 1, comprising: the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody is used for detecting and functionally identifying the Chlamydomonas reinhardtii IFT144 protein.

Technical Field

The invention relates to the field of bioengineering, and particularly relates to a chlamydomonas reinhardtii IFT144 protein polyclonal antibody, and a preparation method and application thereof.

Background

Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) is one of the major model organisms for studying cilia function in eukaryotes. Cilia, also called flagella, are a type of organelle that protrude above the cell surface of eukaryotes and have a polar structure. It was found that there are probably over 600 soluble proteins in cilia, but there are no organelles in cilia required for protein synthesis, such as ribosomes, etc., so that protein molecules required for cilia biosynthesis are transported into and out of cilia by intracollicular transporter (IFT) proteins, which cause abnormal assembly or disaggregation of cilia once IFT proteins are deleted.

In the process of implementing the invention, the inventor finds that the prior art has at least the following problems:

the antibody corresponding to the IFT protein is one of important materials required for researching the function of the corresponding protein, the types of the antibodies corresponding to the IFT protein are very few at present, and the specificity and the titer of the existing antibodies are not high, which brings inconvenience for researching the function of the IFT protein and is not beneficial to researching the pathogenesis of cilia-related diseases and preventing the diseases.

Disclosure of Invention

In order to solve the problems in the prior art, the embodiment of the invention provides a chlamydomonas reinhardtii IFT144 protein polyclonal antibody, and a preparation method and application thereof. The technical scheme is as follows:

in one aspect, the embodiment of the invention provides a Chlamydomonas reinhardtii IFT144 protein polyclonal antibody, wherein the amino acid sequence of the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody is shown as SEQ ID NO. 1 in a sequence table.

In another aspect, the embodiment of the present invention provides a method for preparing the chlamydomonas reinhardtii IFT144 protein polyclonal antibody, where the method includes:

constructing a recombinant expression vector, wherein the nucleotide sequence of the recombinant expression vector comprises a sequence shown as SEQ ID NO. 2 in a sequence table;

transforming the recombinant expression vector into a competent cell to obtain a recombinant expression strain;

inducing the protein expression of the recombinant expression strain to obtain Chlamydomonas reinhardtii IFT144 protein;

and taking the Chlamydomonas reinhardtii IFT144 protein as an antigen, and performing immune treatment to obtain the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody.

Specifically, the method for constructing the recombinant expression vector comprises the following steps: extracting total RNA of chlamydomonas reinhardtii;

performing reverse transcription by taking the total RNA of the chlamydomonas reinhardtii as a template to obtain chlamydomonas reinhardtii cDNA;

performing PCR amplification by using the Chlamydomonas reinhardtii cDNA as a template to obtain a Chlamydomonas reinhardtii IFT144 target gene;

and connecting the chlamydomonas reinhardtii IFT144 target gene with a prokaryotic expression vector to obtain a recombinant expression vector.

Further, the prokaryotic expression vector is a double-enzyme-digestion Escherichia coli expression vector pET-28 a.

Further, an upstream primer and a downstream primer are adopted for PCR amplification, the sequence of the upstream primer is shown as SEQ ID NO. 3 in the sequence table, and the sequence of the downstream primer is shown as SEQ ID NO. 4 in the sequence table.

Further, the method for inducing protein expression of the recombinant expression strain comprises the following steps: culturing the recombinant expression strain at 37 ℃ overnight, then transferring the recombinant expression strain into a small shake flask of a fresh LB liquid culture medium containing 50 mu g/mL kanamycin to prepare a bacterial liquid, and when the concentration of the bacterial liquid reaches OD₆₀₀And when the concentration is 0.6-0.8, adding isopropyl- β -D-thiogalactoside with the final concentration of 0.1-1 mM for induction, wherein the induction temperature is 37 ℃, and the induction time is 3h, so as to obtain the Chlamydomonas reinhardtii IFT144 protein.

Specifically, the method further comprises: purifying the obtained chlamydomonas reinhardtii IFT144 protein; the purification method comprises the following steps: performing first centrifugation on the obtained chlamydomonas reinhardtii IFT144 protein to obtain first centrifugal precipitate;

carrying out second centrifugation after the first centrifugal precipitation is re-suspended and washed by PBS (phosphate buffer solution), so as to obtain second centrifugal precipitation;

re-suspending the second centrifugal precipitate by using the PBS, and adding lysozyme to obtain a re-suspension;

adding PMSF into the heavy suspension, performing ultrasonic treatment, adding Triton X-100 and DTT, and centrifuging to obtain a precipitate;

performing denaturation and dissolution on the precipitate by using a denaturation solution to obtain a denaturation solution;

diluting and renaturing the denatured solution by using renaturation solution to obtain renaturation solution;

and carrying out column chromatography separation on the renaturation solution, and eluting to obtain the purified Chlamydomonas reinhardtii IFT144 protein.

Specifically, the method for the immune treatment comprises the following steps: mixing the chlamydomonas reinhardtii IFT144 protein antigen with complete Freund's adjuvant to obtain a first mixed solution, injecting the first mixed solution into an immune animal body, mixing the IFT144 protein antigen with the incomplete Freund's adjuvant after 12 days to obtain a second mixed solution, injecting the second mixed solution into the immune animal body, mixing the IFT144 protein antigen with the incomplete Freund's adjuvant after 14 days to obtain a third mixed solution, injecting the third mixed solution into the immune animal body, mixing the IFT144 protein antigen with the incomplete Freund's adjuvant after 14 days to obtain a fourth mixed solution, and injecting the fourth mixed solution into the immune animal body.

Specifically, the immune animal is a New Zealand white rabbit.

In another aspect, the embodiment of the present invention provides an application of the chlamydomonas reinhardtii IFT144 protein polyclonal antibody, where the application includes: the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody is used for detecting and functionally identifying the Chlamydomonas reinhardtii IFT144 protein.

The technical scheme provided by the embodiment of the invention has the following beneficial effects: the embodiment of the invention provides a Chlamydomonas reinhardtii IFT144 protein polyclonal antibody, a preparation method and an application thereof, wherein the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody can specifically recognize Chlamydomonas reinhardtii IFT144 protein and has the characteristics of high titer and good specificity; the preparation method obtains a cDNA sequence of the IFT144 protein of the Chlamydomonas reinhardtii through PCR amplification cloning, and obtains an antigen for preparing an antibody through in vitro expression and purification by constructing a recombinant expression vector; the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody is used for detecting and functionally identifying Chlamydomonas reinhardtii IFT144 protein.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a SDS-PAGE electrophoresis of proteins according to the present invention, wherein the ordinate represents the standard molecular weight of the proteins; 1 in the abscissa is 4mg/mL BSA; 2 is Marker; 3, ultrasonic supernatant; 4 is inclusion body washing supernatant; 5, 2-fold dilution for inclusion bodies; 6 is the inclusion body diluted by 5 times; 7, diluting the inclusion body by 10 times;

FIG. 2 is an SDS-PAGE electrophoresis of purified inclusion body protein provided in the example of the present invention, wherein Marker is the protein standard molecular weight; 1 is Marker; 2 is 4mg/mL BSA; 3, diluting the purified inclusion body protein by 5 times; 4, diluting the inclusion body protein by 10 times after purification;

FIG. 3 is a graph of antibody titer assessed by ELISA provided in the examples of the present invention, wherein the abscissa is different dilution ratios; ordinate is OD₄₅₀An absorption value;

FIG. 4 is a Westernblot assay result of a polyclonal antibody against Chlamydomonas reinhardtii IFT144 protein according to an embodiment of the present invention, in which the ordinate on the left side is labeled as the protein standard molecular weight;

FIG. 5 is a diagram showing the results of the Westernblot assay of flagellin obtained by isolating flagella from a polyclonal antibody against Chlamydomonas reinhardtii IFT144 protein and Chlamydomonas reinhardtii 21gr, wherein the left ordinate is labeled as the protein standard molecular weight.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.

The embodiment of the invention provides a Chlamydomonas reinhardtii IFT144 protein polyclonal antibody, wherein the amino acid sequence of the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody is shown as SEQ ID NO. 1 in a sequence table.

The preparation method of the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody is briefly described as follows, and comprises the following steps:

constructing a recombinant expression vector, wherein the nucleotide sequence of the recombinant expression vector comprises a sequence shown as SEQ ID NO. 2 in a sequence table;

transforming the recombinant expression vector into a competent cell to obtain a recombinant expression strain;

inducing the protein expression of the recombinant expression strain to obtain Chlamydomonas reinhardtii IFT144 protein;

and taking the Chlamydomonas reinhardtii IFT144 protein as an antigen, and performing immune treatment to obtain the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody.

Specifically, the method for constructing the recombinant expression vector comprises the following steps: extracting total RNA of chlamydomonas reinhardtii;

and carrying out reverse transcription by taking the total RNA of the chlamydomonas reinhardtii as a template to obtain chlamydomonas reinhardtii cDNA.

And performing PCR amplification by using the Chlamydomonas reinhardtii cDNA as a template to obtain the Chlamydomonas reinhardtii IFT144 target gene.

Connecting the Chlamydomonas reinhardtii IFT144 target gene with a prokaryotic expression vector to obtain a recombinant expression vector

Specifically, the prokaryotic expression vector is a double-enzyme-digestion Escherichia coli expression vector pET-28 a.

Specifically, an upstream primer and a downstream primer are used for amplification during PCR amplification, the sequence of the upstream primer is shown as SEQ ID NO. 3 in a sequence table, and the sequence of the downstream primer is shown as SEQ ID NO. 4 in the sequence table.

Specifically, the method for inducing the protein expression of the recombinant expression strain comprises the following steps: culturing the recombinant expression strain at 37 ℃ overnight, then transferring the recombinant expression strain into a small shake flask of a fresh LB liquid culture medium containing 50 mu g/mL kanamycin to prepare a bacterial liquid, and when the concentration of the bacterial liquid reaches OD₆₀₀And when the concentration is 0.6-0.8, adding isopropyl- β -D-thiogalactoside with the final concentration of 0.1-1 mM for induction, wherein the induction temperature is 37 ℃, and the induction time is 3h, so as to obtain the Chlamydomonas reinhardtii IFT144 protein.

Specifically, the competent cell is an escherichia coli competent cell.

Further, the competent cells were escherichia coli Rosetta competent cells.

Specifically, the method further comprises: purifying the obtained chlamydomonas reinhardtii IFT144 protein; the purification method comprises the following steps: performing first centrifugation on the obtained chlamydomonas reinhardtii IFT144 protein to obtain first centrifugal precipitate;

carrying out second centrifugation after the first centrifugal precipitation is re-suspended and washed by PBS (phosphate buffer solution), so as to obtain second centrifugal precipitation;

re-suspending the second centrifugal precipitate by using the PBS, and adding lysozyme to obtain a re-suspension;

adding PMSF into the heavy suspension, performing ultrasonic treatment, adding Triton X-100 and DTT, and centrifuging to obtain a precipitate;

performing denaturation and dissolution on the precipitate by using a denaturation solution to obtain a denaturation solution;

diluting and renaturing the denatured solution by using renaturation solution to obtain renaturation solution;

and carrying out column chromatography separation on the renaturation solution, and eluting to obtain the purified Chlamydomonas reinhardtii IFT144 protein.

Specifically, the method for immunological treatment comprises the following steps: mixing Chlamydomonas reinhardtii IFT144 protein antigen with complete Freund's adjuvant to obtain a first mixed solution, injecting the first mixed solution into an immune animal body, mixing the IFT144 protein antigen with the incomplete Freund's adjuvant after 12 days to obtain a second mixed solution, injecting the second mixed solution into the immune animal body, mixing the IFT144 protein antigen with the incomplete Freund's adjuvant after 14 days to obtain a third mixed solution, injecting the third mixed solution into the immune animal body, mixing the IFT144 protein antigen with the incomplete Freund's adjuvant after 14 days to obtain a fourth mixed solution, and injecting the fourth mixed solution into the immune animal body.

Further, the immunized animal is a new zealand white rabbit.

The application of the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody comprises the step of applying the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody to the detection and functional identification of the Chlamydomonas reinhardtii IFT144 protein.

In this example, the method of constructing the recombinant expression vector comprises:

the total RNA of the chlamydomonas reinhardtii is extracted to obtain the total RNA of the chlamydomonas reinhardtii, and the total RNA of the chlamydomonas reinhardtii is extracted by adopting a general plant total RNA extraction kit provided by Beijing Baitach biotechnology limited, and the extraction method refers to the instruction of the kit.

And performing reverse transcription by taking the total RNA of the chlamydomonas reinhardtii as a template to obtain the cDNA of the chlamydomonas reinhardtii.

The Chlamydomonas reinhardtii cDNA is used as a template, and PCR (Polymerase Chain Reaction) amplification is carried out to obtain the Chlamydomonas reinhardtii IFT144 target gene.

During PCR amplification, an upstream primer and a downstream primer are adopted for amplification, and the amplification procedure is pre-denaturation at 95 ℃ for 5 min; (denaturation 95 ℃, 30 s; annealing 60 ℃, 30 s; extension 72 ℃, 1min) x 35 cycles; extension was 72 ℃ for 5 min. The upstream primer is 5'-AGCAAATGGGTCGCGGATCCATGGCCC GGGAGCGG-3', the sequence of the upstream primer is shown as SEQ ID NO. 3 in the sequence table, and GGATCC in the upstream primer is a BamHI enzyme cutting site; the downstream primer is 5'-TTGTCGACGGAGCTCGAATTCTTACACCGCAGCCTGCGTC-3', the downstream primer is shown as SEQ ID NO. 4 in the sequence table, and GAATTC in the downstream primer is an EcoRI enzyme cutting site.

And carrying out agarose gel electrophoresis analysis and identification on the target gene of Chlamydomonas reinhardtii IFT144 and an Escherichia coli expression vector pET-28a subjected to double enzyme digestion by BamH I enzyme and EcoRI enzyme, cutting the gel and recovering the gel to obtain a recovered PCR product and a recovered Escherichia coli expression vector pET-28 a.

And connecting the recovered PCR product to the BamH I and EcoRI sites of the recovered Escherichia coli expression vector pET-28a by using homologous recombinase to obtain a recombinant expression vector, and reacting at 37 ℃ for 30 min. Transforming the recombinant expression vector into an escherichia coli competent cell (the escherichia coli competent cell is an escherichia coli Rosetta competent cell) to obtain a recombinant expression strain containing the chlamydomonas reinhardtii IFT144 gene coding sequence, culturing the recombinant expression strain on an LB solid plate containing 100 mu g/mL kanamycin at 37 ℃ for 15h, picking a single colony on the LB solid plate, carrying out PCR (polymerase chain reaction) identification on the single colony, and then carrying out sequencing analysis, wherein the sequencing result shows that the obtained coding region of the target gene (chlamydomonas reinhardtii IFT144) is consistent with the expected coding region, and the nucleotide sequence of the coding region of the target gene (chlamydomonas reinhardtii IFT144) is shown as SEQ ID NO:2 in a sequence table.

Culturing the recombinant expression strain at 37 ℃ overnight, then transferring the recombinant expression strain into a 50mL small shake flask containing a fresh LB liquid culture medium containing 50 mu g/mL kanamycin to prepare a bacterial liquid, and when the concentration of the bacterial liquid reaches OD₆₀₀When the concentration is 0.6-0.8, IPTG (isopropyl- β -D-thiogalactose) with the final concentration of 0.1-1 mM is addedGlycoside), inducing the protein expression of the recombinant expression strain, wherein the induction temperature is 37 ℃, and the induction time is 3 h. Centrifuging to remove supernatant, adding 1 xSDS boiling water, boiling for 10min, taking a small amount of induced Chlamydomonas reinhardtii IFT144 protein, performing SDS-PAGE electrophoretic analysis, obtaining good induction result, and enlarging the culture volume of bacteria to obtain a large amount of Chlamydomonas reinhardtii IFT144 protein.

Purifying the obtained chlamydomonas reinhardtii IFT144 protein. And (3) centrifugally collecting the induced recombinant expression strain after the expression of the Chlamydomonas reinhardtii IFT144 protein, centrifuging for 10min at 2800rpm at 4 ℃ to obtain a first centrifugal precipitate, carrying out heavy suspension and washing on the first centrifugal precipitate by PBS (Phosphate Buffer Saline), and then centrifuging again, wherein the centrifugation conditions are the same as the above conditions to obtain a second centrifugal precipitate. And (3) freezing the collected recombinant expression strain (precipitate obtained by secondary centrifugation) in liquid nitrogen, resuspending the bacteria by using PBS, resuspending the bacteria by using 30-40 mL of PBS by using 1L of bacterial liquid, then adding lysozyme with the final concentration of 1mg/mL, uniformly mixing, and standing on ice for 30-60 min to obtain a resuspension solution. PMSF (phenylmethylsulfonyl fluoride) with the final concentration of 1mM is added into the heavy suspension and transferred into a small beaker, the small beaker is placed in a container containing ice, then the heavy suspension is crushed by ultrasound, the container containing the ice can keep the ultrasonic process at a low temperature, so that the protein degradation is prevented, the ultrasonic crushing time and power are determined according to specific conditions, when the heavy suspension is crushed by ultrasound in the embodiment, the work time is 7s, the pause time is 5s, the power is 30%, the ultrasonic crushing time is 8min, and the heavy suspension is crushed by ultrasound until the heavy suspension is clear and bright. After completion of ultrasonication, Triton X-100 and 1mM DTT (dithiothreitol) were added to the small beaker at final concentrations of 1%, respectively, and then the small beaker was left on ice for 30 min. Then, the mixture was centrifuged at 10000rpm at 4 ℃ for 60min to obtain a supernatant and a precipitate. Washing the collected precipitate, performing denaturation and dissolution by PBS (Phosphate buffered Saline) to dissolve inclusion bodies, wherein the PBS comprises 8mol/L urea and 20mmol/L DTT, diluting the dissolved inclusion bodies by a renaturation solution (the solvent in the renaturation solution is PBS and comprises 0.5mmol/L EDTA, 5% glycerol and 1mmol/L DTT) until the final concentration of the urea is 0.5mol/L to obtain a renaturation solution, performing column chromatography separation on the renaturation solution to specifically combine the protein with beads, eluting the protein, converting the protein solution into the PBS, allowing the concentration of the purified protein to reach more than 1mg/mL, performing electrophoresis on the purified protein, detecting the purity of the protein and estimating the concentration of the protein, the purified protein was stored at-80 ℃. SDS-PAGE was performed, and the expression results of the purified proteins were analyzed, and as shown in FIGS. 1 and 2, it can be seen from FIG. 1 that the target protein (recombinant Chlamydomonas reinhardtii IFT144) was distributed in inclusion bodies after induction expression, and the inclusion body proteins were selected and purified. As can be seen from FIG. 2, the purified inclusion body protein (recombinant Chlamydomonas reinhardtii IFT144) has a small amount of impurity bands, but the purity meets the requirement of immunization, and the measured concentration is about 5mg/mL, so that the inclusion body protein can be used for immunization.

The purified recombinant Chlamydomonas reinhardtii IFT144 protein is used as an antigen to immunize New Zealand white rabbits. 1mL of blood was collected from the ear vein of a New Zealand white rabbit, and the mixture was allowed to stand overnight at 4 ℃ to collect the supernatant as a negative control. 0.6mg of the purified recombinant Chlamydomonas reinhardtii IFT144 protein is taken, then an equal volume of Freund complete adjuvant is added, the mixture is uniformly mixed and emulsified by an injector until the mixture does not diffuse in water, and then the subcutaneous immunization is carried out at six points on the back and one point is carried out at each subcutaneous immunization of double hind feet. The time of the first immunization was recorded. After 12 days, a second immunization was performed, 0.3mg of recombinant Chlamydomonas reinhardtii IFT144 protein was mixed with an equal volume of Freund's incomplete adjuvant and emulsified until no diffusion in water, and four points were subcutaneously immunized on the back.

Two weeks later, a third immunization was performed, and 0.3mg of recombinant Chlamydomonas reinhardtii IFT144 protein was directly immunized subcutaneously at the back for four spots. And at this time, 1ml of blood was taken from the marginal vein of the ear, and the serum antibody titer was preliminarily measured.

Two weeks later, a fourth immunization was performed, and 0.3mg of recombinant Chlamydomonas reinhardtii IFT144 protein was directly immunized to the back at four spots subcutaneously.

After 12 days, taking about 80mL of whole blood, anesthetizing, taking blood from carotid until death, refrigerating the whole blood at 4 ℃ overnight, separating upper serum to obtain the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody, detecting the titer of the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody in the serum, and subpackaging and storing at-80 ℃. The antibody titer is determined by an ELISA method by taking the negative serum as a negative control, the highest dilution factor of the diluted antibody serum when the absorbance at 450nm is 2.1 times greater than that of the serum before immunization is defined as the antibody titer, the result of the ELISA method is shown in figure 3, and the prepared Chlamydomonas reinhardtii IFT144 polyclonal antibody antiserum titer is about 1:512000 as can be seen from figure 3; the specificity of the antibody is determined by Westernblot, the determination results are respectively shown in FIG. 4 and FIG. 5, and it can be seen from FIG. 4 and FIG. 5 that the prepared Chlamydomonas reinhardtii IFT144 polyclonal antibody can be specifically combined with the recombinant Chlamydomonas reinhardtii IFT144 protein and the flagellin obtained by separating flagella from Chlamydomonas reinhardtii 21gr, and the size of the formed band is consistent with the prediction, which indicates that the specificity of the obtained antibody is good.

The embodiment of the invention provides a Chlamydomonas reinhardtii IFT144 protein polyclonal antibody, a preparation method and an application thereof, wherein the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody can specifically recognize Chlamydomonas reinhardtii IFT144 protein and has the characteristics of high titer and good specificity; the preparation method obtains a cDNA sequence of the IFT144 protein of the Chlamydomonas reinhardtii through PCR amplification cloning, and obtains an antigen for preparing an antibody through in vitro expression and purification by constructing a recombinant expression vector; the Chlamydomonas reinhardtii IFT144 protein polyclonal antibody is used for detecting and functionally identifying Chlamydomonas reinhardtii IFT144 protein.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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<120> chlamydomonas reinhardtii IFT144 protein polyclonal antibody, and preparation method and application thereof

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Met Ala Ala Gly Ala Leu Leu Thr Pro Leu Val Gly Leu His Thr Leu

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Leu Ile Val Ala Ala Ala Thr Ala Leu Ala Ala Ile Met Ala Leu Ala

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Gly Gly Ala Leu Thr Val Thr Leu Thr Ala Gly Val Thr Gly Ala Thr

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Ile Leu Thr Pro His Leu Leu Ala Val Pro Met Ala Leu Cys Val Ser

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Ala Leu Leu Gly Ala Gly Thr Ser Ala Ala Gly Gly Ala Thr Thr Ser

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Leu Ala Ile Ala Ala Leu Thr Leu Thr Ile Met Gly Cys Thr Ala Gly

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Gly Ala Ala Pro Leu Gly Leu Ala Pro Leu Ala Thr Thr Gly Pro Ile

100 105 110

Met Leu His Ser Thr Pro Gly Ala Gly Thr Ile Leu Leu Gly Thr Leu

115 120 125

Ala Gly Thr Val Ala Val Val Ser Ser His Ser Ala Gly Ile Ser Gly

130 135 140

Gly Val His Ser Gly Leu Thr Leu Ala Thr Leu Thr Ala Val Thr Thr

145 150 155 160

Cys Ala Ser Leu Gly AlaVal Ala Met Ala Gly Ala Ala Cys Val Ala

165 170 175

Val Leu Ala Ala Ala Ala Ala Thr Ala Gly Ile Leu Gly Ala Ala Val

180 185 190

Ala Leu Ala Ala Ala Gly Ala Ile Gly Leu Val Gly Thr Thr Leu Ala

195 200 205

Gly Gly Val Leu Thr Val Gly Thr His Ala Gly Thr Met His Ser Pro

210 215 220

Leu Ala Ser Leu Pro Met Val Thr Ala Pro His Gly Thr Ala Val Leu

225 230 235 240

Thr Leu Thr Ser Leu Leu Gly Met Thr Leu Leu Ala Val Ser Ala Ala

245 250 255

Gly Thr Val Ala Ala Ile Gly Leu Gly Ala Gly Pro Ala Pro Cys Gly

260 265 270

Leu Gly Pro Ser His Ala Ala Val Gly Met Ala Ala Gly Ala Ala Pro

275 280 285

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

290 295 300

Leu Gly Thr Ile Thr Ala Ile Leu Leu Ala Gly Thr Gly Ala Ala Val

305 310 315 320

<210>2

<211>957

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

gcccgggagc ggaagaagac gccgctggtg ggcaagcaca cgaagaagat tgtggctgcc 60

gcctggaaca aggacaacat catggcgctg gcggggcagg acaagaccgt cacgctgacg 120

gacggcgtca cgggcgacac catcaaaact ttccacctca aggacgtgcc catggacctg 180

tgcgtgtctg acaagaagga ggacggctac tcgcggcgag aggagaacac ctactcgctc 240

aacatcaatc ggaaaacgct gtacatcatg cagtgcactg ccgagggcga ccggccgctg 300

gagctggcgt tcctggacac ctacggcccc atcatgaagc acagctggtt cggcgacggc 360

tacattctgc tgggctacaa gaacggctac gtggcggtgg tgtccagtca cagccgcgag 420

atcagcgagg aggtgcactc cgggaagtac ctggacacgt tgacggacgt gacctactgc 480

gccagcctgg gccgcgtggc catggcgggc gccaactgcg tgcgtgtgct ggacgccaac 540

gccgactaca acgagatcaa gggcgacgcg gtggacctgg acgcaaacca ggccatcgag 600

aaggtcggct ggaccaagga cggccaggtg ctgacggtgg gcacgcacaa tggctacatg 660

cattcgttcc tggcctcgct acccatggtg tacgacttcc acggcacccg cgtgctgtac 720

ctgaccagct tgctggagat gacgctgctg gacgtgagcc ggcgccagac cgtggcgcgc 780

attgagctgg agaacgagcc ggcgttctgc gggctgggcc cctcacacgc ggcggtgggc 840

atgaacaacc aggccgcgtt ctacagcctg ggcgagaagg tgggcaaggt ggtgcagcgg 900

cgcgagtacc tgggcaccat caccgccatc aagctcaacg agacgcaggc tgcggtg 957

<210>3

<211>35

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

agcaaatggg tcgcggatcc atggcccggg agcgg 35

<210>4

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ttgtcgacgg agctcgaatt cttacaccgc agcctgcgtc 40