Recombinant R7 protein of leucocyte worm Ka, preparation method and application thereof
阅读说明：本技术 卡氏住白细胞虫重组r7蛋白及其制备方法和应用 (Recombinant R7 protein of leucocyte worm Ka, preparation method and application thereof ) 是由 邝春曼 谭志坚 黄仪娟 刘丽丹 翁亚彪 王新秋 于 2019-09-02 设计创作，主要内容包括：本发明属于生物技术领域,公开了一种卡氏住白细胞虫重组R7蛋白及其制备方法；所述卡氏住白细胞虫重组R7蛋白具有的氨基酸序列为MBP-R7。本发明人工合成了卡氏住白细胞虫重组R7基因及制备卡氏住白细胞虫重组R7蛋白,表达产物经纯化、体外活性检测后发现卡氏住白细胞虫重组R7蛋白具有检测卡氏住白细胞虫抗体的作用,在检测卡氏住白细胞虫病中,用重组R7蛋白代替第二代裂殖体作为包被抗原,检测效果更好,更快速便捷；由于重组R7蛋白更易制备,因此降低了抗原获取的难度和成本。(The invention belongs to the technical field of biology, and discloses a recombinant R7 protein of Ka shi leucocyte worm and a preparation method thereof; the recombinant Leucocyte caris karst R7 protein has an amino acid sequence of MBP-R7. The recombinant R7 gene of the Ka-type leucocyte worm is artificially synthesized, the recombinant R7 protein of the Ka-type leucocyte worm is prepared, an expression product is purified and subjected to in vitro activity detection, and then the recombinant R7 protein of the Ka-type leucocyte worm has the function of detecting the antibody of the Ka-type leucocyte worm, and in the detection of the Ka-type leucocyte worm, the recombinant R7 protein is used for replacing a second generation schizont as a coating antigen, so that the detection effect is better, and the detection is quicker and more convenient; because the recombinant R7 protein is easier to prepare, the difficulty and cost of obtaining the antigen are reduced.)
1. The recombinant Karldown leucocyte R7 protein is characterized in that the amino acid sequence of the recombinant Karldown leucocyte R7 protein is MBP-R7.
2. The method of producing recombinant leukocyte kahn R7 protein of claim 1, comprising the steps of:
(1) carrying out PCR amplification on the gene R7 of the Kaschin leucocyte to obtain a recombinant R7 gene of the Kaschin leucocyte;
(2) carrying out enzyme digestion on the psYNO-1 plasmid by using restriction endonuclease, connecting a recombinant R7 gene of the Kaschin leucocyte with the enzyme-digested psYNO-1 plasmid by using ligase, transforming the connected plasmid into E.coli TOP10 competent cells for culture, carrying out PCR identification positive cloning to obtain PCR identification positive bacterial liquid, extracting the plasmid of the PCR identification positive bacterial liquid, and carrying out double enzyme digestion identification to obtain a positive recombinant plasmid psYNO-R7;
(3) transforming the positive recombinant plasmid psYNO-R7 into E.coli BL21 expression bacteria, and adding isopropyl thiogalactoside for induction expression to obtain psYNO-R7 recombinant protein;
(4) and purifying the psYNO-R7 recombinant protein after induction expression to obtain the recombinant R7 protein of the Kascherma leucocytozoon.
3. The method of claim 2, wherein the recombinant leukocyte depletion R7 gene is derived from a leukocyte depletion-resistant Karl-leucocyte R7 gene comprising the steps of:
(1) finding out a nucleotide sequence of the leucocyte R7, and synthesizing an R7 gene sequence after codon optimization;
(2) designing a pair of nucleotide sequences of the specific connection primers as follows: CTGTACTTCCAGGGAGCAAGTGGTCTGGTTACC and GTGGTGGTGCTCGAGTTATCACACTTCTTCATGT, and designing a pair of identifying primers with nucleotide sequences of GACTAATTCGAGCTCGAACAACAACA and CATGTTCTTCTTTTTCTTTCTCTTCGTG;
(3) and (3) taking the synthesized R7 gene as a template, and carrying out PCR amplification on the R7 gene fragment to obtain the recombinant R7 gene of the Karschner leucocytozoon.
4. The method for producing recombinant R7 Ka leukocyte cell protein according to claim 3, wherein the 5 'end of the specific ligation primer contains 15 bases homologous to the end of the expression plasmid vector psYNO-1 and the 3' end contains a R7 gene-specific primer sequence.
5. The method according to claim 2, wherein in the step (2), the restriction enzymes are BamHI and XhoI.
6. The preparation method of claim 5, wherein in the step (2), the double-enzyme digestion identification step is as follows: psYNO-R7, SacI, XhoI, 10 XM Buffer and ddH2And adding the O into a sterilized 1.5mL centrifuge tube, uniformly mixing, carrying out constant-temperature water bath for 2h at 37 ℃ to obtain a digestion product, carrying out agarose gel electrophoresis on the digestion product, marking the plasmid identified as positive, sequencing to obtain a positive recombinant plasmid psYNO-R7, and storing at-20 ℃.
7. The use of the recombinant karyoblastic leucocyto-sis R7 protein of claim 1 for detecting karyoblastic leucosis.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant R7 protein of Ka's leucocyte and a preparation method thereof.
Background
The chicken Kashi leucocytozoonosis is also called white crown disease and is a kind of blood sporozoosis caused by the fact that the Kashi leucocytozoonosis is parasitized in red blood cells, white blood cells and visceral tissue cells of chicken. The chicken Ka's leucocytozoonosis mostly occurs in chicks of 3-6 weeks, the disease is serious, and the death rate is high; the infection rate of young chickens is higher than that of chicks, but the death rate is not high; adult chickens have the highest infection rate but have low mortality rate and mild symptoms. The main clinical symptoms are cockscomb pallor, emaciation, white or green thin manure in a water drawing sample, the development of chickens is hindered, the egg production of adult chickens is reduced or even stopped, and great economic loss can be caused to the chicken industry.
The primary judgment of the Ka's leucocytozoosis can be made according to the diagnosis methods of clinical symptoms, epidemiology, pathological anatomy, etc., and the confirmation of the diagnosis generally adopts a blood smear examination method to examine whether merozoites or gametocytes exist in peripheral blood. The volume of merozoites appearing in peripheral blood is extremely small, and the merozoites of the Kashi leucocytozoon and other haemosporidium are difficult to distinguish under a common optical microscope, so misdiagnosis is easy. And the appearance time of the mature gametophyte of the Kashi leucocyte in peripheral blood is late and short, and the blood smear examination is easy to miss. Although simple and easy, the blood smear inspection method has great limitations in the aspects of detection efficiency, detection rate, insect species identification and the like, is not more and more suitable for the requirements of current prevention and treatment work, and particularly cannot be suitable for large-scale epidemiological investigation.
The detection of the chicken Ka-Gu-Bai-Ka-Bai-Ka-: namely specificity, proportionality, reversibility and stage property. Therefore, it is desirable to provide a recombinant leukocyte cell karst R7 protein for detecting recombinant leukocyte cell karst antibodies.
Disclosure of Invention
The invention aims to provide a recombinant Ka-leucocyte R7 protein and a preparation method thereof, and the recombinant Ka-leucocyte R7 protein replaces the second generation schizont of the Ka-leucocyte to be used as a coating antigen, so that the preparation is easy and the cost is low.
In order to achieve the purpose, the invention adopts the technical scheme that:
the inventors coated the ELISA plate with the prokaryotic expression recombinant R7 protein to establish an ELISA method for detecting anti-Karl-leucocytozoon antibodies. The R7 protein is an outer membrane antigen of the second-generation schizont of the Leucocyte Karlobis. Compared with an agar diffusion test, a blood smear examination method and the like, the ELISA detection method based on the recombinant R7 protein of the Kashi leucocytozoon is more sensitive, convenient and rapid, and can detect the antibody generated after the chicken is infected with the Kashi leucocyte and the antibody generated after the vaccine of the recombinant R7 oil adjuvant is inoculated. Provides help for the prevention and control of the Kashi leucocytozoonosis in the vast farms in China.
The recombinant Karldown leucocyte R7 protein has an amino acid sequence of MBP-R7.
The MBP-R7:
MGHHHHHHGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGENLYFQGASGLVTFISPNNVQAEIINTHGVRCNQNEEVTHQTHQTHQTHQTHQTHQTHQIHQIHQIHGYMTNQKHEEHGKIINQVKENVKNTVNENVKNNVDENTTSEHEITIPNENDIKTNDENETTHYEREIIYIVDDLPEVNVEESDETEHITYEIDNDIQEEHEKVTHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEIEKEEHEEVIHEEEKEEVTHEEKEKEEHEEV。
a preparation method of recombinant R7 protein of leucocyte caterpillar comprises the following steps:
(1) carrying out PCR amplification on the gene R7 of the Kaschin leucocyte to obtain a recombinant R7 gene of the Kaschin leucocyte;
(2) carrying out enzyme digestion on the psYNO-1 plasmid by using restriction endonuclease, connecting a recombinant Ka leucocyte R7 gene with the enzyme-digested psYNO-1 plasmid by using ligase, transforming the connected plasmid into E.coli TOP10 competent cells for culture, carrying out PCR identification positive cloning to obtain PCR identification positive bacterial liquid, extracting the plasmid of the PCR identification positive bacterial liquid, and carrying out double enzyme digestion identification to obtain a positive recombinant plasmid psYNO-R7;
(3) transforming the positive recombinant plasmid psYNO-R7 into E.coli BL21 expression bacteria, and adding isopropyl thiogalactoside for induction expression to obtain psYNO-R7 recombinant protein;
(4) and purifying the psYNO-R7 recombinant protein after induction expression to obtain the recombinant R7 protein of the Kascherma leucocytozoon.
Preferably, the preparation method of the recombinant R7 gene of the Karl-leucocyte worm comprises the following steps:
(1) finding out the gene sequence of the Ka-shi leucocyte worm R7, and artificially synthesizing the R7 gene sequence after codon optimization;
(2) designing a pair of nucleotide sequences of the specific connection primers as follows: CTGTACTTCCAGGGAGCAAGTGGTCTGGTTACC and GTGGTGGTGCTCGAGTTATCACACTTCTTCATGT, and designing a pair of identifying primers with nucleotide sequences of GACTAATTCGAGCTCGAACAACAACA and CATGTTCTTCTTTTTCTTTCTCTTCGTG;
(3) and (3) taking the synthesized R7 gene as a template, and carrying out PCR amplification on the R7 gene fragment to obtain the recombinant R7 gene of the Karschner leucocytozoon.
Preferably, the 5 'end of the specific ligation primer contains 15 bases homologous to the end of the expression plasmid vector psYNO-1 and the 3' end contains the R7 gene specific primer sequence.
Preferably, in step (2), the restriction enzymes are BamHI and XhoI.
Preferably, in step (2), the double enzyme digestion identification step is: psYNO-R7, SacI, XhoI, 10 XMbuffer and ddH2Adding O into a sterilized 1.5mL centrifuge tube, mixing uniformly, carrying out constant-temperature water bath at 37 ℃ for 2h to obtain a restriction enzyme product, carrying out agarose gel electrophoresis on the restriction enzyme product, marking the plasmid identified as positive, and sequencing to obtain a positive recombinant plasmid psYNO-R7, and preservingStored at-20 ℃.
Preferably, in step (2), the step of identifying positive clones by PCR is: carrying out PCR reaction on the bacterial liquid, wherein the PCR reaction system comprises Ex Taq DNA polymerase, primers, bacterial liquid and ddH2O。
More preferably, the primer is: R7S2-F and R7S 2-R; the Ex Taq DNA polymerase, the primer, the bacterial liquid and the ddH2The volume of O is respectively: 25 μ L, 2 μ L, 4 μ L and 19 μ L.
More preferably, Ex Taq DNA polymerase is a product of TaKaRa.
The PCR amplification program of the bacterial liquid comprises the following steps:
the reaction conditions are pre-denaturation at 94 ℃ for 1min, (denaturation at 94 ℃ for 20s, annealing at 60 ℃ for 20s, and extension at 72 ℃ for 30s) x 34 cycles, extension at 72 ℃ for 5min, and finishing at 4 ℃.
Carrying out electrophoresis on the obtained PCR product by using 1% agarose gel, carrying out electrophoresis at 135V for 25min, observing, photographing and recording the result; and (3) identifying positive bacteria liquid by PCR, uniformly mixing 500 mu L of bacteria liquid with 500 mu L of 50% glycerol, marking, and storing at-20 ℃.
Preferably, in step (2), the double enzyme digestion identification step is: psYNO-R7, SacI, XhoI, 10 XMbuffer and ddH2And adding the O into a sterilized 1.5mL centrifuge tube, uniformly mixing, carrying out constant-temperature water bath for 2h at 37 ℃ to obtain a digestion product, carrying out agarose gel electrophoresis on the digestion product, marking the plasmid identified as positive, sequencing to obtain a positive recombinant plasmid psYNO-R7, and storing at-20 ℃.
More preferably, the psYNO-R7, SacI, XhoI, 10 XM Buffer and ddH2The volumes of O were 25. mu.L, 1. mu.L, 5. mu.L and 18. mu.L, respectively.
An application of recombinant R7 protein of Ka-shi leucocyte in detecting Ka-shi leucocytozoonosis.
An indirect ELISA detection method adopting recombinant R7 protein of the leucocytozoon casseliflavus, which comprises the following steps:
(1) coating antigen: diluting the recombinant R7 protein of the leucocyte worm Ka to 0.065-1.3 mug/mL by using a coating buffer solution, and coating for 1-2h at 37 ℃ or overnight at 4 ℃;
(2) washing the enzyme label plate: spin-drying the coating liquid in the pores, adding a phosphate Tween buffer solution for washing, and spin-drying the residual liquid;
(3) and (3) sealing: adding sealing liquid, sealing at 37 deg.C for 1-2h or sealing at 4 deg.C overnight;
(4) adding serum: diluting the serum with 5% skimmed milk, mixing and standing for 5-10min, adding diluted serum into an enzyme label plate, and incubating at 37 deg.C for 0.5-2 h;
(5) adding enzyme-labeled secondary antibody: adding diluted goat anti-chicken IgG labeled with HRP, and incubating at 37 ℃ for 0.5-2 h;
(6) color development: adding TMB color developing solution, and developing at 37 deg.C for 10-30 min;
(7) and (4) terminating: adding a stop solution to stop color development;
(8) reading: and detecting the OD value of the 450nm wavelength by using a microplate reader, and reading.
Preferably, in step (1), the coating buffer is selected from carbonate buffer selected from 0.2mol/L NaHCO at pH 9.63。
Preferably, in step (2), the phosphate tween buffer (PBST) is a phosphate buffer containing 0.05% tween 20 and having a pH of 7.2.
Preferably, after the step (3) and before the step (4), after the step (4) and before the step (5), after the step (5) and before the step (6), the method further comprises the steps of spin-drying the liquid in the pores, adding a phosphate Tween buffer solution for washing, and spin-drying the residual liquid.
Preferably, in the step (3), the sealing liquid is 5% skimmed milk powder;
preferably, in step (4), the dilution ratio of the serum to 5% skim milk is 1:250 to 1: 2000. More preferably, the serum is diluted to 5% skim milk at a 1:500 dilution ratio.
Preferably, in the step (5), the dilution ratio of the HRP-labeled goat anti-chicken IgG is 1:2000-1: 12000. More preferably, the dilution ratio of the HRP-labeled goat anti-chicken IgG is 1: 4000.
Preferably, in the step (6), the color developing solution is SureBlue TMB MicrowellSubstrate from KPL company, the substrate is 3,3',5,5' -tetramethylbenzidine, and the reaction time of the color developing solution is 10-30 min.
Preferably, in the step (7), the stop solution is 2mol/L H2SO4。
The beneficial technical effects of the invention are as follows:
the recombinant R7 gene of the Ka-type leucocyte worm is artificially synthesized, the recombinant R7 protein of the Ka-type leucocyte worm is prepared, an expression product is purified and subjected to in vitro activity detection, and then the recombinant R7 protein of the Ka-type leucocyte worm has the function of detecting the Ka-type leucocyte worm disease, in the detection of the Ka-type leucoworm disease, the recombinant R7 protein is used for replacing a second generation schizont to serve as a coating antigen, so that the detection effect is better, and the detection is quicker and more convenient; because the recombinant R7 protein is easier to prepare, the difficulty and cost of obtaining the antigen are reduced.
Drawings
FIG. 1: l. calleleryi R7 gene PCR amplification result graph;
FIG. 2: a PCR identification result chart of the bacterial liquid;
FIG. 3: a double-enzyme digestion result chart of the recombinant plasmid psYNO-R7;
FIG. 4: an induction time analysis chart;
FIG. 5: IPTG induction concentration analysis plot;
FIG. 6: detecting the presence of the expression product;
FIG. 7: r7 protein purification results;
FIG. 8: detecting the result of Western blot by using R7 protein;
FIG. 9: analyzing the reactogenicity of the recombinant R7 protein;
FIG. 10: protein concentration standard curve.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail by examples below. It should be understood, however, that the description herein of specific embodiments is only intended to illustrate the invention and not to limit the scope of the invention.