CN113307852A - Induced expression and purification method for 47kDa protein of orientia tsutsutsugamushi - Google Patents
Induced expression and purification method for 47kDa protein of orientia tsutsutsugamushi Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, in particular to an inducible expression and purification method for 47kDa protein of tsutsugamushi disease eastern body, which solves the problem that no better method for inducing in vitro and expressing a large amount of related protein of tsutsutsugamushi disease eastern body and adaptive conditions exist in the prior art, and the inducible expression and purification method for 47kDa protein of tsutsugamushi disease eastern body comprises the steps of primer synthesis, acquisition and amplification of target gene, construction of recombinant expression plasmid, inducible expression of related protein of tsutsutsugamushi disease eastern body, Western blot test, analysis of recombinant protein expression form, purification of recombinant protein and the like. The invention designs a method for obtaining 47kDa protein of Oriental tsutsugamushi disease, which successfully induces and expresses 47kDa protein related to Oriental tsutsugamushi disease in vitro by exploring and adjusting protein induced expression conditions and protein purification method, and simultaneously improves the purity of the protein in vitro to obtain the purity of more than 85 percent.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to an inducible expression and purification method for 47kDa protein of Oriental tsutsugamushi.
Background
Tsutsugamushi disease is a natural epidemic disease, also called as jungle typhus, a potentially fatal infectious disease easily overlooked caused by the obligate intracellular bacterium tsutsutsugamushi oriental disease, more than 100 ten thousand cases of tsutsugamushi occur each year, and researches have found that if tsugamushi disease is not treated in time, the fatality rate is likely to be higher than that of dengue fever to reach 10 percent, and especially when a patient is not treated by antibiotics, the fatality rate of the disease can even reach 40 to 45 percent.
Orientia tsutsugamushi parasitizes the tsutsugamushi mite, and after the person is bitten by the tsutsugamushi mite, the Orientia tsutsugamushi grows and breeds in the endothelial cells of blood vessels of the human body, the incubation period is generally 7-21 days, the clinical manifestations mainly include nonspecific symptoms such as chills, fever and the like, and severe infections represent infective shock, disseminated intravascular coagulation and even death. The non-specific symptoms of tsutsugamushi disease at the initial stage of the disease are difficult to be distinguished from other febrile diseases such as fever with thrombocytopenia syndrome, epidemic hemorrhagic fever and the like, so the current research report on the related pathogenic mechanism of tsutsugamushi disease is almost still in a blank field. Although PCR and serological detection and the treatment of sensitive antibiotics have been developed with the progress of research, the research of rapid and accurate diagnosis methods and vaccines with high sensitivity and specificity still has important significance.
The 47kDa protein of Orientia tsutsutsugamushi (Cluster typhus) belongs to the HtrA heat shock protein family and is highly conserved, with > 97% homology among different strains of Orientia tsutsugamushi, with good homologous protection and cross protection against 4 heterologous strains in a lethal challenge mouse model, and thus seems to be a good candidate vaccine for the broad prevention of Orientia tsutsugamushi infection. Serum of patients with tsutsugamushi disease has been shown to recognize several antigens of orientia tsutsutsugamushi, whose major outer membrane 56-kDa protein accounts for 10-15% of the total content of rickettsia cell protein. At present, the 47kDa protein of Oriental tsutsugamushi disease has many researchers on the pathogenic mechanism of the body, but how to stabilize in vitro to obtain the high purity Oriental tsutsugamushi disease related protein has been a difficult point, and there is no method and suitable condition for better in vitro induction and large amount expression of Oriental tsutsugamushi disease related protein at present. Based on the above statement, the present invention proposes an inducible expression and purification method for 47kDa protein of Oriental tsutsugamushi.
Disclosure of Invention
The invention aims to solve the problem that no better method and adaptive conditions for inducing and expressing a large amount of related proteins of Oriental tsutsugamushi in vitro exist in the prior art, and provides a method for inducing, expressing and purifying 47kDa proteins of Oriental tsutsugamushi.
A method for inducible expression and purification of 47kDa protein of Oriental tsutsugamushi disease comprises the following steps:
(1) primer synthesis: designing 1-30 primers of related pathogenic proteins of the orientia tsutsutsugamushi by referring to GenBank and a required enzyme cutting site, totaling 30 primers, and synthesizing the full length of the protein sequence by adopting an overlap pcr method;
(2) obtaining and amplifying target genes: synthesizing 47kDa protein of Oriental tsutsutsugamushi, amplifying the full-length sequence of the 47kDa protein through overlapPcr, sequentially carrying out full-length PCR one-round reaction and full-length PCR two-round reaction, after the reaction is finished, carrying out electrophoresis on the full-length PCR two-round product through 1% agarose gel, and recovering a target fragment for later use;
(3) construction of pET30a-47 recombinant expression plasmid: and (3) treating the pET30a plasmid with BamHI-Xhol during PCR amplification, and recovering glue, wherein the recombination reaction system is as follows: 4ul of recovered product in the step (2), 3.5ul of pET30a + carrier and 2.5ul of recombinase are put in a water bath at 50 ℃ for 25min, placed for 2-3 min to cool, subjected to a colony screening experiment, a monoclonal colony is picked, subjected to shake culture for 12-14h, a bacterial liquid is preserved, a plasmid is extracted, and sequencing is carried out, so that a recombinant bacterial liquid is obtained;
(4) inducible expression of 47kDa protein of Orientia tsutsugamushi: performing shake culture on the recombinant bacterium liquid which is subjected to sequencing and is stored at the same time at 37 ℃ and 220r/min until the bacterium liquid D600 is 0.6-0.8, adding 0.2mM IPTG (isopropyl-beta-thiogalactoside) for induction expression, and collecting the bacterium liquid for ultrasonic treatment to obtain a protein sample;
(5) western blot assay: performing SDS-PAGE test by using the protein sample in the step (4) and 12% separation gel, transferring the protein to a nitrocellulose membrane, closing the nitrocellulose membrane by 5% skim milk for 1h, incubating the nitrocellulose membrane overnight by using a mouse-derived His antibody, washing the protein by PBST for 4 times, washing the protein for 5min each time, incubating the protein by using a goat anti-mouse secondary antibody marked by HRP for 1h at 37 ℃, washing the protein by PBST for 4 times, washing the protein by 5min each time, and developing;
(6) analysis of 47kDa recombinant protein expression form of Orientia tsutsugamushi: recovering the recombinant bacteria stored in the step (3), adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, collecting bacterial liquid, carrying out ultrasonic crushing, centrifuging at 4000r/min for 10min, collecting supernatant, carrying out heavy suspension precipitation by PBS (phosphate buffer solution), respectively collecting supernatant and bacterial inclusion bodies to prepare samples, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test by using 12% separation gel;
(7) purifying 47kDa recombinant protein of Orientia tsutsugamushi: recovering a recombinant bacterium related to the 47kDa protein, transferring the recombinant bacterium to a 1L culture medium, carrying out shaking culture at 37 ℃ until the bacterium solution D600 is 0.6-0.8, adding IPTG (isopropyl thiogalactoside) with the concentration of 0.2mM-0.4mM for induction expression, collecting the protein after 4h, purifying by adopting a Ni-IDA affinity chromatography column, preparing a protein sample, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test by using 12% separation gel, and analyzing the purification effect of the protein;
(8) through the process, a large amount of 47kDa recombinant protein of orientia tsutsugamushi with high purity is obtained successfully through in vitro induction expression and purification.
Preferably, the full-length PCR one-round reaction system in the step (2) is 50 ul: ddH was added to the PCR tube in sequence2O38 ul, polymerase (PV 2) 0.5ul, 5X PV2 buffer 10ul,10mM dNTP 1ul, primers 1-30 each 0.5 ul.
Preferably, the full-length PCR one-round reaction amplification conditions in step (2) are as follows: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 25s, annealing at 62 deg.C for 20 s, and extension at 72 deg.C for 45 s, for 25 cycles; extension at 72 ℃ for 1 min.
Preferably, the full-length PCR two-round reaction system in the step (2)50 ul: ddH was added to the PCR tube in sequence2O37.2 ul, polymerase (PV 2) 0.5ul, 5X PV2 buffer 10ul,10mM dNTP 1ul, primer 10.5 ul, primer 300.5 ul.
Preferably, the conditions for the full-length PCR two-round amplification in step (2) are as follows: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 25s, annealing at 62 deg.C for 20 s, and extension at 72 deg.C for 45 s, for 25 cycles; extension at 72 ℃ for 1 min.
The induced expression and purification method for 47kDa protein of Oriental tsutsugamushi disease provided by the invention has the following beneficial effects:
1. the method for induction expression and mass purification of the related proteins of orientia tsutsutsugamushi, which is proposed by the present invention, can successfully induce and mass express 47kDa protein of orientia tsutsutsutsugamushi in vitro, and at the same time, can successfully obtain 47kDa protein of orientia tsutsutsutsugamushi with higher purity in vitro; the obtained 47kDa protein can be used for further preparation of polyclonal antibodies and monoclonal antibodies, and subsequent preparation of related proteins, polyclonal antibodies and monoclonal antibodies, and the protein can be applied to rapid detection of clinical diseases in the later stage, and simultaneously provides targets for research and development of vaccines.
2. The 47kDa recombinant protein of the orientia tsutsutsugamushi is successfully expressed and purified by a prokaryotic expression system, and meanwhile, the invention lays a foundation for the next preparation of monoclonal antibodies and polyclonal antibodies, the rapid detection methods of related ELISA detection methods and other diseases, the research of related vaccines of genetic engineering and the like.
Drawings
FIG. 1 is an SDS-PAGE analysis of protein expression identification in accordance with the first embodiment of the present invention; in the figure: m represents a protein molecular mass standard; 1 represents pET-30a induction; 2 means no induction; 3 represents after induction; 4 represents the supernatant after induced disruption; 5 represents precipitation after induced disruption.
FIG. 2 is an SDS-PAGE analysis of protein purification in accordance with the first embodiment of the present invention; in the figure: m represents a protein molecular mass standard; 1 represents the treated sample after disruption; 2 denotes outflow; 3-4 represents elution.
FIG. 3 is a Western Blot identification and analysis chart of the protein in the first embodiment of the invention; in the figure: m represents a protein molecular mass standard; 1 represents the purified sample.
FIG. 4 is an SDS-PAGE identification analysis chart of the protein in the first embodiment of the invention; in the figure: m represents a protein molecular mass standard; 1 represents 0.5mg/mL BSA; and 2 represents a purified sample.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Example one
The invention provides an inducible expression and purification method for 47kDa protein of Oriental tsutsugamushi, which comprises the following steps:
(1) primer synthesis: designing 1-30 primers of related pathogenic proteins of the orientia tsutsutsugamushi by referring to GenBank and a required enzyme cutting site, totaling 30 primers, and synthesizing the full length of the protein sequence by adopting an overlap pcr method;
(2) obtaining and amplifying target genes: synthesizing 47kDa protein of Oriental tsutsutsugamushi, amplifying the full-length sequence of the 47kDa protein through overlapPcr, and sequentially carrying out full-length PCR one-round reaction and full-length PCR two-round reaction, wherein the full-length PCR one-round reaction system is 50 ul: ddH was added to the PCR tube in sequence2O38 ul, polymerase (PV 2) 0.5ul, 5X PV2 buffer 10ul,10mM dNTP 1ul, primers 1-30 each 0.5 ul; the full-length PCR one-round reaction amplification conditions are as follows: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 25s, annealing at 62 deg.C for 20 s, and extension at 72 deg.C for 45 s, for 25 cycles; extending for 1 min at 72 ℃; the full-length PCR two-round reaction system is 50 ul: ddH was added to the PCR tube in sequence2O37.2 ul, polymerase (PV 2) 0.5ul, 5X PV2 buffer 10ul,10mM dNTP 1ul, primer 10.5 ul, primer 300.5 ul; the full-length PCR two-round amplification conditions are as follows: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 25s, annealing at 62 deg.C for 20 s, and extension at 72 deg.C for 45 s, for 25 cycles; extending for 1 min at 72 ℃; after the reaction is finished, carrying out electrophoresis on the full-length PCR secondary product through 1% agarose gel, and recovering a target fragment for later use;
(3) construction of pET30a-47 recombinant expression plasmid: and (3) treating the pET30a plasmid with BamHI-Xhol during PCR amplification, and recovering glue, wherein the recombination reaction system is as follows: 4ul of recovered product in the step (2), 3.5ul of pET30a + carrier and 2.5ul of recombinase are put in a water bath at 50 ℃ for 25min, placed for 3 min to cool, subjected to a colony screening experiment, a monoclonal colony is picked, subjected to shaking culture for 14h, a bacterial liquid is preserved, plasmids are extracted, and sequencing is carried out, so that a recombinant bacterial liquid is obtained;
(4) inducible expression of 47kDa protein of Orientia tsutsugamushi: carrying out shake culture on the recombinant bacterium liquid which is subjected to sequencing and is stored at the same time at 37 ℃ and 220r/min until the bacterium liquid D600 is 0.8, adding 0.2mM IPTG (isopropyl-beta-thiogalactoside) for induction expression, and collecting the bacterium liquid for ultrasonic treatment to obtain a protein sample;
(5) western blot assay: performing SDS-PAGE test by using the protein sample in the step (4) and 12% separation gel, transferring the protein to a nitrocellulose membrane, closing the nitrocellulose membrane by 5% skim milk for 1h, incubating the nitrocellulose membrane overnight by using a mouse-derived His antibody, washing the protein by PBST for 4 times, washing the protein for 5min each time, incubating the protein by using a goat anti-mouse secondary antibody marked by HRP for 1h at 37 ℃, washing the protein by PBST for 4 times, washing the protein by 5min each time, and developing;
(6) analysis of 47kDa recombinant protein expression form of Orientia tsutsugamushi: recovering the recombinant bacteria stored in the step (3), adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, collecting bacterial liquid, carrying out ultrasonic crushing, centrifuging at 4000r/min for 10min, collecting supernatant, carrying out heavy suspension precipitation by PBS (phosphate buffer solution), respectively collecting supernatant and bacterial inclusion bodies to prepare samples, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test by using 12% separation gel;
(7) purifying 47kDa recombinant protein of Orientia tsutsugamushi: recovering recombinant bacteria related to 47kDa protein, transferring the recombinant bacteria into a 1L culture medium, carrying out shaking culture at 37 ℃ until the bacterial liquid D600 is 0.8, adding 0.4mM IPTG for induction expression, collecting protein after 4h, purifying by using a Ni-IDA affinity chromatography column, preparing a protein sample, carrying out SDS-PAGE test by using 12% separation gel, and analyzing the protein purification effect;
(8) through the process, a large amount of 47kDa recombinant protein of the orientia tsutsugamushi with higher purity is obtained successfully through in vitro induction expression and purification;
(9) amplifying 47kDa protein gene sequence of Orientia tsutsugamushi: performing gel recovery by using two rounds of amplification of primers, wherein the size of a product is basically consistent with that of an expected product (shown in figure 1);
(10) protein induction expression: collecting the induced bacteria liquid, and the results of 12% SDS-PAGE and Western blot show that the recombinant bacteria can see a single band (shown in figure 2);
(11) analysis of 47kDa protein expression form of Orientia tsutsugamushi: supernatant and inclusion bodies after the crushed bacteria are respectively collected, and SDS-PAGE results show that the recombinant bacteria express proteins in the form of inclusion bodies (as shown in figure 3)
(12) Purifying 47kDa protein of Oriental tsutsugamushi: culturing a large amount of recombinant bacteria of related proteins, collecting protein samples, and purifying by using a Ni-IDA affinity chromatography column, wherein the result shows that the 47kDa recombinant protein is successfully purified (figure 4).
Example two
The invention provides an inducible expression and purification method for 47kDa protein of Oriental tsutsugamushi, which comprises the following steps:
(1) primer synthesis: designing 1-30 primers of related pathogenic proteins of the orientia tsutsutsugamushi by referring to GenBank and a required enzyme cutting site, totaling 30 primers, and synthesizing the full length of the protein sequence by adopting an overlap pcr method;
(2) obtaining and amplifying target genes: synthesizing 47kDa protein of Oriental tsutsutsugamushi, amplifying the full-length sequence of the 47kDa protein through overlapPcr, and sequentially carrying out full-length PCR one-round reaction and full-length PCR two-round reaction, wherein the full-length PCR one-round reaction system is 50 ul: ddH was added to the PCR tube in sequence2O38 ul, polymerase (PV 2) 0.5ul, 5X PV2 buffer 10ul,10mM dNTP 1ul, primers 1-30 each 0.5 ul; the full-length PCR one-round reaction amplification conditions are as follows: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 25s, annealing at 62 deg.C for 20 s, and extension at 72 deg.C for 45 s, for 25 cycles; extending for 1 min at 72 ℃; the full-length PCR two-round reaction system is 50 ul: ddH was added to the PCR tube in sequence2O37.2 ul, polymerase (PV 2) 0.5ul, 5X PV2 buffer 10ul,10mM dNTP 1ul, primer 10.5 ul, primer 300.5 ul; the full-length PCR two-round amplification conditions are as follows: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 25s, annealing at 62 deg.C for 20 s, and extension at 72 deg.C for 45 s, for 25 cycles; extending for 1 min at 72 ℃; after the reaction is finished, carrying out electrophoresis on the full-length PCR secondary product through 1% agarose gel, and recovering a target fragment for later use;
(3) construction of pET30a-47 recombinant expression plasmid: and (3) treating the pET30a plasmid with BamHI-Xhol during PCR amplification, and recovering glue, wherein the recombination reaction system is as follows: 4ul of recovered product in the step (2), 3.5ul of pET30a + carrier and 2.5ul of recombinase are put in a water bath at 50 ℃ for 25min, placed for 2.5 min for cooling, subjected to a colony screening experiment, picked single clone colony is subjected to shake culture for 13h, a bacterial liquid is preserved, a plasmid is extracted, and sequencing is carried out, so as to obtain a recombinant bacterial liquid;
(4) inducible expression of 47kDa protein of Orientia tsutsugamushi: carrying out shake culture on the recombinant bacterium liquid which is subjected to sequencing and is stored at the same time at 37 ℃ and 220r/min until the bacterium liquid D600 is 0.7, adding 0.2mM IPTG (isopropyl-beta-thiogalactoside) for induction expression, and collecting the bacterium liquid for ultrasonic treatment to obtain a protein sample;
(5) western blot assay: performing SDS-PAGE test by using the protein sample in the step (4) and 12% separation gel, transferring the protein to a nitrocellulose membrane, closing the nitrocellulose membrane by 5% skim milk for 1h, incubating the nitrocellulose membrane overnight by using a mouse-derived His antibody, washing the protein by PBST for 4 times, washing the protein for 5min each time, incubating the protein by using a goat anti-mouse secondary antibody marked by HRP for 1h at 37 ℃, washing the protein by PBST for 4 times, washing the protein by 5min each time, and developing;
(6) analysis of 47kDa recombinant protein expression form of Orientia tsutsugamushi: recovering the recombinant bacteria stored in the step (3), adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, collecting bacterial liquid, carrying out ultrasonic crushing, centrifuging at 4000r/min for 10min, collecting supernatant, carrying out heavy suspension precipitation by PBS (phosphate buffer solution), respectively collecting supernatant and bacterial inclusion bodies to prepare samples, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test by using 12% separation gel;
(7) purifying 47kDa recombinant protein of Orientia tsutsugamushi: recovering recombinant bacteria related to 47kDa protein, transferring the recombinant bacteria into a 1L culture medium, carrying out shaking culture at 37 ℃ until the bacterial liquid D600 is 0.7, adding 0.3mM IPTG for induction expression, collecting protein after 4h, purifying by using a Ni-IDA affinity chromatography column, preparing a protein sample, carrying out SDS-PAGE test by using 12% separation gel, and analyzing the protein purification effect;
(8) through the process, a large amount of 47kDa recombinant protein of orientia tsutsugamushi with high purity is obtained successfully through in vitro induction expression and purification.
EXAMPLE III
The invention provides an inducible expression and purification method for 47kDa protein of Oriental tsutsugamushi, which comprises the following steps:
(1) primer synthesis: designing 1-30 primers of related pathogenic proteins of the orientia tsutsutsugamushi by referring to GenBank and a required enzyme cutting site, totaling 30 primers, and synthesizing the full length of the protein sequence by adopting an overlap pcr method;
(2) obtaining and amplifying target genes: synthesizing 47kDa protein of Oriental tsutsutsugamushi, amplifying the full-length sequence of the 47kDa protein through overlapPcr, and sequentially carrying out full-length PCR one-round reaction and full-length PCR two-round reaction, wherein the full-length PCR one-round reaction system is 50 ul: ddH was added to the PCR tube in sequence2O38 ul, polymerase (PV 2) 0.5ul, 5X PV2 buffer 10ul,10mM dNTP 1ul, primers 1-30 each 0.5 ul; the full-length PCR one-round reaction amplification conditions are as follows: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 25s, annealing at 62 deg.C for 20 s, and extension at 72 deg.C for 45 s, for 25 cycles; extending for 1 min at 72 ℃; the full-length PCR two-round reaction system is 50 ul: ddH was added to the PCR tube in sequence2O37.2 ul, polymerase (PV 2) 0.5ul, 5X PV2 buffer 10ul,10mM dNTP 1ul, primer 10.5 ul, primer 300.5 ul; the full-length PCR two-round amplification conditions are as follows: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 25s, annealing at 62 deg.C for 20 s, and extension at 72 deg.C for 45 s, for 25 cycles; extending for 1 min at 72 ℃; after the reaction is finished, carrying out electrophoresis on the full-length PCR secondary product through 1% agarose gel, and recovering a target fragment for later use;
(3) construction of pET30a-47 recombinant expression plasmid: and (3) treating the pET30a plasmid with BamHI-Xhol during PCR amplification, and recovering glue, wherein the recombination reaction system is as follows: 4ul of recovered product in the step (2), 3.5ul of pET30a + carrier and 2.5ul of recombinase are put in a water bath at 50 ℃ for 25min, placed for 2 min to cool, subjected to a colony screening experiment, a monoclonal colony is picked, subjected to shaking culture for 12h, a bacterial liquid is preserved, plasmids are extracted, and sequencing is carried out, so that a recombinant bacterial liquid is obtained;
(4) inducible expression of 47kDa protein of Orientia tsutsugamushi: carrying out shake culture on the recombinant bacterium liquid which is subjected to sequencing and is stored at the same time at 37 ℃ and 220r/min until the bacterium liquid D600 is 0.6, adding 0.2mM IPTG (isopropyl-beta-thiogalactoside) for induction expression, and collecting the bacterium liquid for ultrasonic treatment to obtain a protein sample;
(5) western blot assay: performing SDS-PAGE test by using the protein sample in the step (4) and 12% separation gel, transferring the protein to a nitrocellulose membrane, closing the nitrocellulose membrane by 5% skim milk for 1h, incubating the nitrocellulose membrane overnight by using a mouse-derived His antibody, washing the protein by PBST for 4 times, washing the protein for 5min each time, incubating the protein by using a goat anti-mouse secondary antibody marked by HRP for 1h at 37 ℃, washing the protein by PBST for 4 times, washing the protein by 5min each time, and developing;
(6) analysis of 47kDa recombinant protein expression form of Orientia tsutsugamushi: recovering the recombinant bacteria stored in the step (3), adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, collecting bacterial liquid, carrying out ultrasonic crushing, centrifuging at 4000r/min for 10min, collecting supernatant, carrying out heavy suspension precipitation by PBS (phosphate buffer solution), respectively collecting supernatant and bacterial inclusion bodies to prepare samples, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test by using 12% separation gel;
(7) purifying 47kDa recombinant protein of Orientia tsutsugamushi: recovering recombinant bacteria related to 47kDa protein, transferring the recombinant bacteria into 1L culture medium, carrying out shaking culture at 37 ℃ until the bacterial liquid D600 is 0.6, adding 0.2mM IPTG for induction expression, collecting protein after 4h, purifying by using a Ni-IDA affinity chromatography column, preparing a protein sample, carrying out SDS-PAGE test by using 12% separation gel, and analyzing the protein purification effect;
(8) through the process, a large amount of 47kDa recombinant protein of orientia tsutsugamushi with high purity is obtained successfully through in vitro induction expression and purification.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A method for inducible expression and purification of 47kDa protein of Oriental tsutsugamushi disease, comprising the steps of:
(1) primer synthesis: designing 1-30 primers of related pathogenic proteins of the orientia tsutsutsugamushi by referring to GenBank and a required enzyme cutting site, totaling 30 primers, and synthesizing the full length of the protein sequence by adopting an overlap pcr method;
(2) obtaining and amplifying target genes: synthesizing 47kDa protein of Oriental tsutsutsugamushi, amplifying the full-length sequence of the 47kDa protein through overlapPcr, sequentially carrying out full-length PCR one-round reaction and full-length PCR two-round reaction, after the reaction is finished, carrying out electrophoresis on the full-length PCR two-round product through 1% agarose gel, and recovering a target fragment for later use;
(3) construction of pET30a-47 recombinant expression plasmid: and (3) treating the pET30a plasmid with BamHI-Xhol during PCR amplification, and recovering glue, wherein the recombination reaction system is as follows: 4ul of recovered product in the step (2), 3.5ul of pET30a + carrier and 2.5ul of recombinase are put in a water bath at 50 ℃ for 25min, placed for 2-3 min to cool, subjected to a colony screening experiment, a monoclonal colony is picked, subjected to shake culture for 12-14h, a bacterial liquid is preserved, a plasmid is extracted, and sequencing is carried out, so that a recombinant bacterial liquid is obtained;
(4) inducible expression of 47kDa protein of Orientia tsutsugamushi: performing shake culture on the recombinant bacterium liquid which is subjected to sequencing and is stored at the same time at 37 ℃ and 220r/min until the bacterium liquid D600 is 0.6-0.8, adding 0.2mM IPTG (isopropyl-beta-thiogalactoside) for induction expression, and collecting the bacterium liquid for ultrasonic treatment to obtain a protein sample;
(5) western blot assay: performing SDS-PAGE test by using the protein sample in the step (4) and 12% separation gel, transferring the protein to a nitrocellulose membrane, closing the nitrocellulose membrane by 5% skim milk for 1h, incubating the nitrocellulose membrane overnight by using a mouse-derived His antibody, washing the protein by PBST for 4 times, washing the protein for 5min each time, incubating the protein by using a goat anti-mouse secondary antibody marked by HRP for 1h at 37 ℃, washing the protein by PBST for 4 times, washing the protein by 5min each time, and developing;
(6) analysis of 47kDa recombinant protein expression form of Orientia tsutsugamushi: recovering the recombinant bacteria stored in the step (3), adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, collecting bacterial liquid, carrying out ultrasonic crushing, centrifuging at 4000r/min for 10min, collecting supernatant, carrying out heavy suspension precipitation by PBS (phosphate buffer solution), respectively collecting supernatant and bacterial inclusion bodies to prepare samples, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test by using 12% separation gel;
(7) purifying 47kDa recombinant protein of Orientia tsutsugamushi: recovering a recombinant bacterium related to the 47kDa protein, transferring the recombinant bacterium to a 1L culture medium, carrying out shaking culture at 37 ℃ until the bacterium solution D600 is 0.6-0.8, adding IPTG (isopropyl thiogalactoside) with the concentration of 0.2mM-0.4mM for induction expression, collecting the protein after 4h, purifying by adopting a Ni-IDA affinity chromatography column, preparing a protein sample, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test by using 12% separation gel, and analyzing the purification effect of the protein;
(8) through the process, a large amount of 47kDa recombinant protein of orientia tsutsugamushi with high purity is obtained successfully through in vitro induction expression and purification.
2. The method of claim 1The method for inducible expression and purification of 47kDa protein of Orientia tsutsugamushi is characterized in that the full-length PCR reaction system in the step (2) is 50 ul: ddH was added to the PCR tube in sequence2O38 ul, polymerase (PV 2) 0.5ul, 5X PV2 buffer 10ul,10mM dNTP 1ul, primers 1-30 each 0.5 ul.
3. The method for inducible expression and purification of 47kDa protein of Orientia tsutusgamushi as claimed in claim 1, wherein said full length PCR one-round reaction amplification conditions in step (2) are as follows: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 25s, annealing at 62 deg.C for 20 s, and extension at 72 deg.C for 45 s, for 25 cycles; extension at 72 ℃ for 1 min.
4. The method for inducible expression and purification of 47kDa protein of Orientia tsutusgamushi as claimed in claim 1, wherein said full length PCR two-round reaction system in step (2) is 50 ul: ddH was added to the PCR tube in sequence2O37.2 ul, polymerase (PV 2) 0.5ul, 5X PV2 buffer 10ul,10mM dNTP 1ul, primer 10.5 ul, primer 300.5 ul.
5. The method for inducible expression and purification of 47kDa protein of Orientia tsutusgamushi as claimed in claim 1, wherein said two rounds of full length PCR amplification conditions in step (2) are as follows: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 25s, annealing at 62 deg.C for 20 s, and extension at 72 deg.C for 45 s, for 25 cycles; extension at 72 ℃ for 1 min.
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| CN113186304A (en) * | 2021-06-09 | 2021-07-30 | 海南医学院 | Fluorescence isothermal amplification primer, probe, kit and detection method for orientia tsutsutsugamushi nucleic acid |
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