WO2018119626A1 - 中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒 - Google Patents

中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒 Download PDF

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WO2018119626A1
WO2018119626A1 PCT/CN2016/112283 CN2016112283W WO2018119626A1 WO 2018119626 A1 WO2018119626 A1 WO 2018119626A1 CN 2016112283 W CN2016112283 W CN 2016112283W WO 2018119626 A1 WO2018119626 A1 WO 2018119626A1
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ngal
neutrophil gelatinase
associated lipocalin
antibody
kit according
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PCT/CN2016/112283
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French (fr)
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李瑞净
刘俊鹏
刘春燕
钟冬梅
孟媛
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菲鹏生物股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the present invention relates to the field of biotechnology, and in particular to a neutrophil gelatinase-associated lipocalin detection kit.
  • sCr is a reliable renal function marker protein and plays an important role in the diagnosis of AKI; however, due to the above shortcomings, sCr is not an ideal early diagnosis marker protein for AKI.
  • diagnosis of AKI according to the urine output index is often not well and effectively reflects the renal function damage due to factors such as oliguria, diuretic drug use and complicated operation process in some patients after surgery.
  • researchers are constantly looking for early diagnostic markers for AKI.
  • a variety of AKI marker proteins with clinical potential are currently reported, such as neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecules. -1 (kidney injury molec ⁇ Le-1), cystatin C (cystatin C), and interleukin-18 (IL-18).
  • NGAL has a molecular weight of about 25KD and is covalently bound to neutrophil gelatinase. It is a small molecule protein expressed in neutrophils and epithelial cells of some tissues including renal tubules. It has natural resistance to proteases. . NGAL is a growth factor in physiological state, which is mainly involved in the occurrence and growth of early renal epithelium. The expression level is low in epithelial tissues of normal tissues including kidney, lung, stomach and colon. After renal ischemia-reperfusion injury, NGAL is expressed in the proximal convoluted epithelium, and the expression of NGAL in the kidney is significantly increased by renal injury for different reasons and released into urine and plasma.
  • NGAL levels can significantly increase urinary and blood NGAL levels within 24 hours of injury, more than 24 hours earlier than cystatin C levels, and serum creatinine levels rise later. Therefore, by detecting NGAL levels, renal function can be assessed and treated promptly after acute renal impairment occurs to reduce mortality. Therefore, NGAL can be used as a diagnostic marker for acute renal failure, and is a good indicator for evaluating the clinical treatment effect and prognosis of acute renal failure.
  • NGAL measurement related fields include: cardiovascular surgery patients, critically ill persons, pus or hemorrhagic shock, kidney surgery, chronic kidney disease, cardiorenal syndrome, hepatorenal syndrome, venous X-ray contrast agent response and nephrotoxic treatment response .
  • the average concentration of NGAL in the urine of healthy people is 5.3 ng / mL (range 0.7 ⁇ 9.6 ng / mL); the average concentration of NGAL in plasma is 63 ng / mL (range 37 ⁇ 106 ng / mL).
  • the level of NGAL increased sharply after kidney injury.
  • the NGAL concentration in the urine of patients with arbitrarily selected intensive care unit can reach 110 ng/mL ⁇ 40,000 ng / mL; the average concentration of NGAL in plasma is 25 ng / mL ⁇ 3491 ng / mL.
  • NGAL The detection of NGAL was initially performed by western blotting. This method played an important role in the early quantitative study of NGAL. However, due to its shortcomings such as low sensitivity and accuracy, complicated operation and poor repeatability, it is rarely used. Quantitative study of NGAL; Radioimmunoassay (RIA) is established and perfected in laboratory research. It has high sensitivity, specificity and simple operation, and is widely used in the quantification of NGAL, but radioactive substances. Potential contamination and damage to the environment and laboratory personnel. In 2005, BioPorto of Denmark first launched the ELISA commercial kit, which can be used for NGAL detection of serum (plasma) and urine. Its latest ELISA kit can be used for rapid experimental diagnosis.
  • RIA Radioimmunoassay
  • the product was CE certified in 2006 and can be used for acute kidney. Clinical diagnosis and monitoring of functional impairment, but compared to the latex-enhanced immunoturbidimetric assay, which takes a long time and cannot be automated, the company has developed a latex-enhanced immunoturbidimetric kit, but it is expensive.
  • the detection sensitivity of the conventional NGAL detection kit is low.
  • a neutrophil gelatinase-associated lipocalin detection kit comprising:
  • test solution containing latex particles conjugated with an anti-human neutrophil gelatinase-associated lipocalin antibody
  • a standard which is a recombinant neutrophil gelatinase-associated lipocalin with a native conformation.
  • the neutrophil gelatinase-associated lipocalin assay kit uses latex-enhanced turbidimetry to determine the amount of neutrophil gelatinase-associated lipocalin in a sample compared to conventional NGAL assay kits. It has high sensitivity and, in addition, has the advantages of simple operation, good repeatability, and short time consumption. It does not require pretreatment of samples and can be used on an automatic biochemical analyzer.
  • lane 1 is an SDS-PAGE electrophoresis pattern of recombinant NGAL expressed in Example 1, wherein lane 1 is a protein Marker, lane 2 is a sterol loading boiling treatment, and lane 3 is a non-sterol loading boiling treatment;
  • Figure 3 is a graph showing the correlation between the NGAL kit of the present invention and a control kit.
  • the invention discloses a neutrophil gelatinase-associated lipocalin (NGAL) detection kit, which comprises a buffer, a detection solution and a standard.
  • NGAL neutrophil gelatinase-associated lipocalin
  • the buffer solution comprises 20 mmol to 100 mmol of Tris buffer or phosphate buffer, 50 mmol to 500 mmol of inorganic salt, 0.1 wt% to 5 wt% of a stabilizer, and 0.01 wt% to 0.2 wt% of a preservative, and the pH of the buffer is 7.0 to 8.5.
  • the inorganic salt is at least one selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, and potassium sulfate
  • the stabilizer is selected from at least one of calf albumin, gelatin, glycine, sucrose, Tween 20, and Triton X-100.
  • the preservative is selected from at least one of sodium azide and thimerosal.
  • the NGAL detection kit may not include a buffer, and the user may prepare it by himself.
  • the test solution includes latex particles coupled with an anti-human neutrophil gelatinase-associated lipocalin antibody, 20 mmol to 100 mmol of a buffer, 50 mmol to 500 mmol of an inorganic salt, 0.1 to 5 wt% of a stabilizer, and 0.01 wt%. ⁇ 0.2wt% preservative, the pH of the test solution is 7.0-8.5; wherein the buffer is Tris buffer or phosphate buffer, and the inorganic salt is selected from sodium chloride, potassium chloride, magnesium chloride and potassium sulfate.
  • the stabilizer is at least one selected from the group consisting of calf albumin, gelatin, glycine, sucrose, Tween 20, and Triton X-100, and the preservative is selected from at least one of sodium azide and thimerosal.
  • the latex particles to which the anti-human neutrophil gelatinase-associated lipocalin antibody is conjugated have a particle diameter of 80 nm to 220 nm.
  • the concentration of the latex particles to which the anti-human neutrophil gelatinase-associated lipocalin antibody is conjugated is 1 g/L to 5 g/L.
  • the mass ratio of the anti-human neutrophil gelatinase-associated lipocalin antibody to the latex particles is 10 to 200: 1000.
  • the material of the latex particles may be a carboxylated polystyrene latex or an aminated polystyrene latex.
  • the anti-human neutrophil gelatinase-associated lipocalin antibody is a mixture of at least two murine anti-human monoclonal antibodies.
  • the anti-human neutrophil gelatinase-associated lipocalin antibody is selected from at least two of the monoclonal antibody NGAL-3B5, the monoclonal antibody NGAL-2D8, and the monoclonal antibody NGAL-4F6.
  • the monoclonal antibody NGAL-3B5 was secreted by the hybridoma cell line NGAL-3B5.
  • the hybridoma cell line NGAL-3B5, which secretes the monoclonal antibody NGAL-3B5, was deposited at the China Center for Type Culture Collection (CCTCC) on December 14, 2016. Address: Wuhan University, Wuhan, China.
  • the deposit number is CCTCC No: 2016214, classification and naming: hybridoma cell line NGAL-3B5.
  • the monoclonal antibody NGAL-2D8 was secreted by the hybridoma cell line NGAL-2D8.
  • the hybridoma cell line NGAL-2D8, which secretes the monoclonal antibody NGAL-2D8, was deposited at the China Center for Type Culture Collection (CCTCC) on December 14, 2016. Address: Wuhan University, Wuhan, China.
  • the deposit number is CCTCC No: 2016215, classification and naming: hybridoma cell line NGAL-2D8.
  • the monoclonal antibody NGAL-4F6 was secreted by the hybridoma cell line NGAL-4F6.
  • the hybridoma cell line NGAL-4F6, which secretes the monoclonal antibody NGAL-4F6, was deposited at the China Center for Type Culture Collection (CCTCC) on December 14, 2016. Address: Wuhan University, Wuhan, China. For CCTCC No: 2016216, the classification is named: hybridoma cell line NGAL-4F6.
  • the anti-human neutrophil gelatinase-associated lipocalin antibody comprises monoclonal antibody NGAL-3B5, monoclonal antibody NGAL-2D8 and monoclonal antibody NGAL-4F6.
  • the mass ratio of the monoclonal antibody NGAL-3B5, the monoclonal antibody NGAL-2D8 and the monoclonal antibody NGAL-4F6 is 0.5 to 2:0.5 to 2:1 (optimally 1:1:1).
  • latex particles conjugated with an anti-human neutrophil gelatinase-associated lipocalin antibody are formed by binding of an anti-human neutrophil gelatinase-associated lipocalin antibody and latex particles by chemical crosslinking.
  • the standard is a recombinant neutrophil gelatinase-associated lipocalin with a native conformation.
  • a recombinant neutrophil gelatinase-associated lipocalin having a native configuration is:
  • the nucleotide sequence shown in SEQ ID No. 1 was obtained by screening a dominant epitope segment of the NGAL protein.
  • the latex-enhanced immunoturbidimetric method utilizes an antigen-antibody reaction, and is coupled with latex particles by an anti-human neutrophil gelatinase-associated lipocalin antibody to form an antibody-latex complex, when the latex-conjugated antibody is compared with
  • a low-concentration NGAL has a specific immune response
  • the turbidity of the solution can be significantly changed, and the turbidity of the reaction solution is positively correlated with the NGAL content in the sample within a certain range, and the absorbance change of the reaction solution is measured at a certain wavelength to determine the human body.
  • the amount of NGAL in serum/urine is measured by an antigen-antibody reaction, and is coupled with latex particles by an anti-human neutrophil gelatinase-associated lipocalin antibody to form an antibody-latex complex, when the latex-conjugated antibody is compared with
  • the turbidity of the solution can be significantly changed, and the turbidity of the reaction solution
  • the neutrophil gelatinase-associated lipocalin assay kit uses latex-enhanced turbidimetry to determine the amount of neutrophil gelatinase-associated lipocalin in a sample compared to conventional NGAL assay kits. It has high sensitivity and, in addition, has the advantages of simple operation, good repeatability, and short time consumption. It does not require pretreatment of samples and can be used on an automatic biochemical analyzer.
  • NGAL active dominant epitope DNA fragment was artificially synthesized by PCR polymerase amplification.
  • the upstream primer carries the BamHI site
  • the downstream primer carries the EcoRI site and the EcoRI site
  • the PCR fragment is digested with BamHI and EcoRI, and ligated into the expression vector PK2 after digestion with BamHI and EcoRI.
  • the recombinant plasmid PK2-NGAL was obtained, and after sequencing, it was cultured with 500 mL LB containing 100 ⁇ g/mL kanamycin sulfate (Shanghai Shenggong Bioengineering Technology Service Co., Ltd., hereinafter referred to as the laborer, item No. A506636).
  • the mixture was shake-cultured at 37 ° C to an OD600 of about 1.0, and induced with IPTG (Biotech, Cat. No. A100487) at a final concentration of 0.5 mM for 4 hours at 37 °C.
  • the cells were collected by centrifugation at 7000 rpm for 3 minutes at 4 ° C.
  • the cells of each liter of bacteria were resuspended in 20 mL of lysis buffer (50 mM Tirs-HCl, pH 8.0, 500 mM NaCl), sonicated, centrifuged at 12000 g for 4 minutes at 4 ° C, and collected.
  • the NI column was cleaned (Buffer A: 50 mM Tirs-HCl, pH 8.0, 500 mM NaCl; Buffer B: 50 mM Tirs-HCl, pH 8.0, 500 mM NaCl, 200 mM imidazole).
  • the protein was added and the unbound protein was washed away with 10 times the volume of Buffer A, followed by 5% BufferB to remove the heteroprotein, 20% BufferB. Elut the protein of interest.
  • the target protein was supplemented with 5 mM EDTA, and the protein concentration was measured, and subjected to SDS-PAGE electrophoresis to obtain Fig. 1.
  • the recombinant NGAL antigen has a natural configuration, contains dimers and multimers, and can be used as a standard for NGAL detection.
  • the resulting recombinant NGAL antigen was stored at -20 ° C until use.
  • the recombinant NGAL was diluted to 1.0 mg/mL, mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, product number: F5881), and fully emulsified to obtain an oily emulsion.
  • the emulsion was subcutaneously administered to BALB/c mice at a dose of 0.2 mL (Guangdong Medical Laboratory Animal Center: No. 119, Huangqiyang Road, Nanhai, Foshan City, Guangdong province, 6-week-old female, 5) back sites.
  • the abdominal cavity was boosted, that is, the same amount of antigen was mixed with the Freund's incomplete adjuvant (Sigma-Aldrich, F5506) in the same volume, and the immunization was boosted to four needles.
  • the tail blood was collected and the serum was separated for indirect ELISA.
  • the titer is determined by the method, and the titer is higher than 1:10000 for fusion.
  • BALB/c mouse peritoneal macrophages were used as feeder cells.
  • the BALB/c mice were sacrificed by neck-stretching, 75% alcohol was immersed in the whole body, and the abdominal skin was cut with scissors under aseptic operation.
  • the peritoneum was exposed, and 5 mL of RPMI 1640 basic culture solution was injected into the abdominal cavity with a syringe. Rinse repeatedly, recover the rinse solution, centrifuge at 1000 rpm for 5 minutes, leave a precipitate, resuspend with RPMI 1640 screening medium (in RPMI 1640 complete medium containing HAT), adjust the cell concentration to 1 ⁇ 10 5 /mL, and add 96 wells. Plates, 150 ⁇ L/well, 37 ° C, 5% CO 2 were incubated overnight.
  • mice Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a dish, rinsed once with RPMI 1640 base medium, and placed in a nylon beaker on a small beaker to be filtered to prepare a cell suspension. After centrifugation, the supernatant was discarded, and the RPMI 1640 base medium was resuspended, and this was repeated three times and counted.
  • Mouse myeloma cells Sp2/0 (preserved by Fei Peng Biotechnology Co., Ltd.) were screened by 8-azaguanine and cultured to logarithmic growth phase. Two large bottles were prepared to make cell suspension, centrifuged, and the supernatant was discarded. RPMI1640 was used. The base medium was resuspended and repeated three times, counting.
  • the myeloma cells and the immune spleen cells were mixed at a ratio of 1:10, and washed once with a RPMI 1640 base culture solution in a 50 mL plastic centrifuge tube, and centrifuged at 1200 rpm for 8 minutes. The supernatant was discarded, the cells were mixed, 1 mL of 50% PEG1500 fusion was slowly added, and after 1 minute of fusion, 15 mL of RPMI1640 basal medium was added to terminate the cell fusion. Centrifuge for 5 minutes at 1000 rpm.
  • the supernatant was discarded, and gently incubated with 50 mL of RPMI 1640 screening medium, aliquoted into 10 96-well plates plated with feeder cells, 50 ⁇ L/well, and cultured at 37 ° C, 5% CO 2 . The culture was continued until the sixth day, and the HT medium (HTMI-containing RPMI1640 complete medium) was changed twice.
  • the PK2-NGAL protein was diluted with 0.06 M pH 9.6 carbonate buffer solution to a final concentration of 2 ⁇ g/mL. 0.1 mL per well was added to a 96-well polystyrene plate and incubated at 37 ° C for 2 hours or 4 ° C overnight. The next day, it was blocked with 0.02 M pH 7.2 PBS containing 10% calf serum or 1% skim milk powder at 0.15 mL/well for 2 hours at 37 ° C for detection.
  • mice Six to eight weeks of robust BALB/c mice were selected, and each mouse was intraperitoneally injected with 0.5 mL of pristane; after 10 days, 1 x 10 6 hybridoma cells were intraperitoneally injected. After inoculation of cells for 7 to 10 days, ascites can be produced, and the animal's health and ascites signs should be closely observed. As much as possible of ascites, while the mice are killed before death, the mice are sacrificed and the ascites is inhaled into the test tube with a dropper. The mice can obtain 5-10 mL of ascites. The ascites was collected, centrifuged, and the supernatant was diluted with 3 volumes of PBS and filtered through a filter paper.
  • the resulting filtrate was applied to a protein G affinity chromatography column (GE) equilibrated with PBS at a flow rate of 1 mL/min.
  • the material not adsorbed by protein G was then washed with PBS at a flow rate of 1 mL/min until the adsorption value at OD280 nm reached the baseline.
  • the antibody was eluted with 0.1 M glycine eluate (pH 2.5) and recovered.
  • the recovered antibody was immediately neutralized with 0.1 M Tris (pH 8.8) and the concentration was measured by running a gel.
  • the above-mentioned secretory antibody against human NGAL hybridoma has an antibody titer of 2.77 ⁇ 10 6 or more.
  • the purified monoclonal antibody to be identified was diluted with 0.06 M pH 9.6 carbonate buffer solution to a final concentration of 1 ⁇ g/mL. 0.1 mL per well was added to a 96-well polystyrene plate and incubated at 37 ° C for 2 hours or 4 ° C overnight. The next day, 0.02 M pH 7.2 PBS containing 10% calf serum or 1% skim milk powder, 0.15 mL/well, blocked at 37 °C for 2 hours, and 2000-fold diluted horseradish peroxidase-labeled NGAL epitope was added to identify the antigen.
  • the buffer is 25 mM Tris + 25 mM glycine + 0.1% BSA + 50 mM NaCl + 0.5% Tween 20 + 0.1% NaN3 + 25mMMgCl 2+ 0.1% EDTA, pH 8.2;
  • the chemical and cross-linking methods were used to link the antibody to the latex, and one monoclonal antibody was mixed on average in different epitopes.
  • the monoclonal antibodies secreted by the hybridoma cell line NGAL-3B5, the hybridoma cell line NGAL-2D8 and the hybridoma cell line NGAL-4F6 were mixed at a mass ratio of 1:1:1. ,Proceed as follows:
  • Blocking adding an equal amount of 0.5M glycine + 2% BSA, blocking at pH 8.237 degrees for 1 h or 4 degrees overnight, and centrifuging at 20000 rpm for 30 min;
  • NGAL kit and the control kit of the present invention an internationally renowned company NGAL kit
  • 50 human serums were measured using a Mindray BS-480 automatic biochemical analyzer, and correlation analysis was performed on the measured values. image 3.
  • the NGAL kit was subjected to a 37 degree 3 day, 7 days, and 10 days heat burst test.
  • the data results are shown in Table 2 and Table 3.

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Abstract

一种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，包括：检测液，所述检测液中含有偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒；标准品，所述标准品为具有天然构型的重组中性粒细胞明胶酶相关脂质运载蛋白。

Description

中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒 技术领域：
本发明涉及生物技术领域，特别是涉及一种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒。
背景技术：
在过去的几十年里人们对急性肾损伤(AKI)发病机理的认识和医疗技术水平都有了很大的提高，但AKI的发病率和病死率仍然居高不下。导致这一局面的一个主要原因是缺乏有效的早期诊断标志物。以血清肌酐(Serum creatinine，sCr)升高或排尿量为指标的RIFLE(Rise，Injury，Failure，Loss，End-stage renal disease)诊断系统是目前临床实践对AKI判定的标准方法。但由于受年龄、性别、肌肉的质量、肌肉新陈代谢、药物使用及水合作用等因素的影响，sCr水平变化很大。另一个事实是sCr水平往往在AKI发生几天后才有显著升高。因此，sCr虽然是可靠的肾功能标志蛋白并在AKI的诊断过程中起了重要的作用；但由于以上缺点，sCr不是理想的AKI早期诊断标志蛋白。另外根据排尿量指标进行AKI诊断常常由于手术后一些病人的少尿症、利尿药物的使用及复杂操作过程等因素的影响而不能很好的、有效的反映肾功能损伤情况。科研工作者不断寻找AKI早期诊断标志物，目前多种具有临床应用潜力的AKI标志蛋白被相继报导，如中性粒细胞明胶酶相关脂质运载蛋白(NGAL，neutrophil gelatinase-associatedlipocalin)、肾损伤分子-1(kidney injury molecμLe-1)、半胱氨酸蛋白酶抑制剂C(cystatin C)及白细胞介素-18(IL-18)等。2003年Devarjan，P.课题组在急性缺血再灌注和顺铂诱导的的急性肾损伤小鼠模型中，发现在损伤发生数小时内即可检测到NGAL的的表达明显升高。并且研究者发现NGAL升高较早期的肾脏损 伤的其他指标出现的要早，此后，NGAL在肾损伤早期诊断中潜在价值越来越受到人们重视。
NGAL分子量约为25KD，共价结合于中性粒细胞明胶酶，是在包括肾小管在内的一些组织器官的中性粒细胞和上皮细胞中表达的小分子蛋白，对蛋白酶有天然的抗性。NGAL生理状态下是一种生长因子，主要参与了早期肾脏上皮的发生、生长。在正常组织包括肾脏，肺，胃及结肠的上皮组织中表达量较低。肾脏缺血再灌注损伤后，近曲小管上皮大量表达NGAL，且NGAL在肾脏中的表达会因不同原因的肾损伤而显著升高，并释放到尿液和血浆中。NGAL水平可在损伤的两个小时内，尿液和血液中NGAL水平将显著增加，比胱抑素C水平的升高提早24小时以上，而血清肌酐水平的升高则更晚。因此通过检测NGAL水平有助于在急性肾功能损伤发生后立即对肾功能进行评估并做出及时的处理，以降低死亡率。因此NGAL可作为急性肾衰的诊断标记物，是有效评价急性肾衰临床治疗效果及预后的良好指标。
通常50％以上的重症监护的病人都有不同程度的肾损伤，急性肾功能衰竭死亡率处于危重病死亡率前列。在过去的40年中，对急性肾衰竭的预测并没有得到明显发展，目前诊断方法只能针对肾衰竭中后期，如血清肌酐或胱蛋白酶抑制剂C，只在肾功能恶化后才有所表达，而这可能在器官损伤1天以上才显现甚至仍不明显。以此为指标来诊断肾损伤，指导临床用药，错过了肾损伤最佳治疗窗口，导致病人花费大幅增加、预后不良、发展成慢性肾病甚至死亡。另外NGAL测定相关领域有：心血管手术患者、病危人员、脓血性或出血性休克、肾脏手术、慢性肾病、心肾综合征、肝肾综合征、静脉X射线造影剂的反应和肾毒性治疗反映。
研究表明：健康人群尿液中NGAL平均浓度为5.3ng/mL(范围0.7～9.6ng/mL)；血浆中NGAL平均浓度63ng/mL(范围37～106ng/mL)。肾损伤后NGAL水平急剧上升。任意选择的重症监护室患者尿液中NGAL浓度可达到110ng/mL～40000ng/mL；血浆中NGAL平均浓度25ng/mL～3491ng/mL。尿NGAL水平高于350ng/mL或血浆NGAL水平高于400ng/mL，急性肾功能 衰竭阳性预测值约为90％。
NGAL的检测最初采用western blotting法，此方法在早期NGAL的定量研究过程中起过很重要的作用，但由于该方法灵敏度和精确性低、操作复杂、重复性差等缺点，现在已很少用于NGAL的定量研究；放射免疫测定法(Radioimmunoassay，RIA)是在实验室研究中建立和完善的，该方法具有灵敏度高、特异性强、操作简单等特点而广泛应用于NGAL的定量，但放射性物质可能对环境和实验人员造成潜在污染和伤害。2005年丹麦BioPorto公司首先推出ELISA商品试剂盒，可用于血清(浆)、尿液的NGAL检测，其最新ELISA试剂盒可用于快速实验诊断，该产品于2006年获得欧盟CE认证，可用于急性肾功能损伤的临床诊断和监测，但相对于胶乳增强免疫比浊检测法来说，耗时长，不能实现自动化，后该公司又研发出胶乳增强免疫比浊试剂盒，但价格昂贵。
综上，目前传统的NGAL检测试剂盒的检测灵敏度较低。
发明内容：
基于此，有必要提供一种检测灵敏度较高的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒。
一种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，包括：
检测液，所述检测液中含有偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒；及
标准品，所述标准品为具有天然构型的重组中性粒细胞明胶酶相关脂质运载蛋白。
这种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒采用胶乳增强比浊法来测定样品中中性粒细胞明胶酶相关脂质运载蛋白的含量，相对于传统的NGAL检测试剂盒，检测灵敏度较高，此外，还具有操作简单、重复性好、耗时短等优点，不需要预处理样品且能够在全自动生化分析仪上使用。
附图说明
图1为实施例1中表达得到的重组NGAL的SDS-PAGE电泳图，其中，泳道1为蛋白Marker，泳道2为有巯醇loading煮沸处理，泳道3为无巯醇loading煮沸处理；
图2为实施例3中得到的NGAL的校准曲线；
图3为本发明的NGAL试剂盒与对照试剂盒的相关性对比图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明不受下面公开的具体实施的限制。
本发明公开了一种中性粒细胞明胶酶相关脂质运载蛋白(NGAL)检测试剂盒，包括缓冲液、检测液和标准品。
缓冲液包括20mmol～100mmol的Tris缓冲液或磷酸盐缓冲液、50mmol～500mmol无机盐、0.1wt％～5wt％的稳定剂和0.01wt％～0.2wt％防腐剂，缓冲液的pH为7.0～8.5；其中，无机盐选自氯化钠、氯化钾、氯化镁和硫酸钾中的至少一种，稳定剂选自小牛白蛋白、明胶、甘氨酸、蔗糖、吐温20和TritonX-100中的至少一种，防腐剂选自叠氮钠和硫柳汞中的至少一种。
在其他的实施例中，上述NGAL检测试剂盒也可以不包括缓冲液，使用者自行配制即可。
检测液包括偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒、20mmol～100mmol的缓冲液、50mmol～500mmol无机盐、0.1wt％～5wt％的稳定剂和0.01wt％～0.2wt％防腐剂，检测液的pH为7.0～8.5；其中，缓冲液为Tris缓冲液或磷酸盐缓冲，无机盐选自氯化钠、氯化钾、氯化镁和硫酸钾 中的至少一种，稳定剂选自小牛白蛋白、明胶、甘氨酸、蔗糖、吐温20和TritonX-100中的至少一种，防腐剂选自叠氮钠和硫柳汞中的至少一种。
优选的，偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒的粒径为80nm～220nm。
优选的，检测液中，偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒的浓度为1g/L～5g/L。
优选的，偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒中，抗人中性粒细胞明胶酶相关脂质运载蛋白抗体和胶乳颗粒的质量比为10～200∶1000。
优选的，胶乳颗粒的材料可以是羧基化的聚苯乙烯胶乳或氨基化的聚苯乙烯胶乳。
优选的，抗人中性粒细胞明胶酶相关脂质运载蛋白抗体为至少两种鼠抗人单克隆抗体形成的混合物。
具体来说，抗人中性粒细胞明胶酶相关脂质运载蛋白抗体选自单克隆抗体NGAL-3B5、单克隆抗体NGAL-2D8和单克隆抗体NGAL-4F6中的至少两种。
单克隆抗体NGAL-3B5为杂交瘤细胞株NGAL-3B5分泌得到。可分泌单克隆抗体NGAL-3B5的杂交瘤细胞株NGAL-3B5于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：2016214，分类命名：杂交瘤细胞株NGAL-3B5。
单克隆抗体NGAL-2D8为杂交瘤细胞株NGAL-2D8分泌得到。可分泌单克隆抗体NGAL-2D8的杂交瘤细胞株NGAL-2D8于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：2016215，分类命名：杂交瘤细胞株NGAL-2D8。
单克隆抗体NGAL-4F6为杂交瘤细胞株NGAL-4F6分泌得到。可分泌单克隆抗体NGAL-4F6的杂交瘤细胞株NGAL-4F6于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号 为CCTCC No：2016216，分类命名：杂交瘤细胞株NGAL-4F6。
特别优选的，抗人中性粒细胞明胶酶相关脂质运载蛋白抗体包括单克隆抗体NGAL-3B5、单克隆抗体NGAL-2D8和单克隆抗体NGAL-4F6。其中，单克隆抗体NGAL-3B5、单克隆抗体NGAL-2D8和单克隆抗体NGAL-4F6的质量比为0.5～2∶0.5～2∶1(最优为1∶1∶1)。
一般来说，偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒为抗人中性粒细胞明胶酶相关脂质运载蛋白抗体和胶乳颗粒通过化学交联法结合形成。
标准品为具有天然构型的重组中性粒细胞明胶酶相关脂质运载蛋白。
具体来说，具有天然构型的重组中性粒细胞明胶酶相关脂质运载蛋白为：
(a)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸编码得到的多肽；
(b)、与SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或者
(c)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。
SEQ ID No.1所示的核苷酸序列为筛选NGAL蛋白的优势表位区段得到。
胶乳增强免疫比浊法是利用抗原抗体反应，通过抗人中性粒细胞明胶酶相关脂质运载蛋白抗体与胶乳颗粒偶联，形成抗体-胶乳的复合物，当偶联了胶乳的抗体与较低浓度的NGAL发生特异性的免疫反应时，能够显著改变溶液的浊度，并在一定范围内反应液浊度与样本中NGAL含量呈正相关，在一定波长处测定反应液吸光度变化，从而测定人体血清/尿液中NGAL的含量。
这种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒采用胶乳增强比浊法来测定样品中中性粒细胞明胶酶相关脂质运载蛋白的含量，相对于传统的NGAL检测试剂盒，检测灵敏度较高，此外，还具有操作简单、重复性好、耗时短等优点，不需要预处理样品且能够在全自动生化分析仪上使用。
以下为具体实施例。
实施例中采用药物和仪器如非特别说明，均为本领域常规选择。实施例中未注明具体条件的实验方法，通常按照常规条件，例如文献、书本中所述的条件或者试剂盒生产厂家推荐的方法实现。
实施例1、免疫源和标准品的制备。
采用基因工程技术手段，通过大量的分子生物学分析软件分析筛选出NGAL蛋白的优势表位基因区段，并对其进行大肠杆菌优势密码子优化，序列为SEQ ID No.1，设计overlap PCR引物，通过PCR聚合酶扩增的方式人工合成NGAL活性优势表位DNA片段。其上游引物带有BamHI位点，下游引物带有EcoRI位点且EcoRI位点，PCR的片段经回收用BamHI和EcoRI酶切，连接到用BamHI和EcoRI酶切之后的表达载体PK2(菲鹏生物股份有限公司)中，得到重组质粒PK2-NGAL，经测序正确后用含100μg/mL硫酸卡那霉素(上海生工生物工程技术服务有限公司，以下简称生工，货号A506636)的500mL LB培养基37℃振荡培养至OD600＝1.0左右，用终浓度为0.5mM的IPTG(生工，货号A100487)37℃诱导4小时。4℃7000rpm离心3分钟收集菌体，每升菌液的菌体用20mL裂解缓冲液(50mM Tirs-HCl，pH8.0，500mM NaCl)重悬，超声破碎，4℃12000g离心20分钟，收集上清过NI柱(BufferA：50mM Tirs-HCl，pH8.0，500mM NaCl；BufferB：50mM Tirs-HCl，pH8.0，500mM NaCl，200mM咪唑)。用10倍柱床体积的Buffer A平衡Ni-NTA亲和柱之后，加入蛋白样，用10倍介质体积的Buffer A洗去未结合的蛋白，之后用5％BufferB洗去杂蛋白，20％BufferB洗脱目的蛋白。目的蛋白补加5mM EDTA后测定蛋白浓度，进行SDS-PAGE电泳，得到图1。
由图1可以看出，重组NGAL抗原具有天然构型，含二聚体及多聚体，可以作为NGAL检测的标准品。将得到的重组NGAL抗原-20℃保存备用。
实施例2、抗人NGAL杂交瘤细胞株的建立及其单克隆抗体的制备
1.重组NGAL免疫小鼠
将重组NGAL稀释到1.0mg/mL，与弗氏完全佐剂(Sigma-Aldrich公司，货号：F5881)等体积混合，并充分乳化，得到油状乳液。将该乳液以0.2mL的剂量皮下施给BALB/c小鼠(广东省医学实验动物中心：广东省佛山市南海黄岐鄱阳路119号，6周龄雌性，5只)背部位点。第一次免疫14天后腹腔增强免疫，即等量抗原与弗氏不完全佐剂(Sigma-Aldrich公司，F5506)等体积混合，增强免疫到四针后，采尾血，分离血清，用间接ELISA法测定效价，效价高于1∶10000即可用于融合。
融合前3天，用相同剂量抗原与等体积0.9％氯化钠注射液混合腹腔注射追加免疫，免疫方法同上。
2.杂交瘤细胞系的制备
(1)饲养细胞的制备
以BALB/c鼠腹腔巨噬细胞作饲养细胞。在融合前1天，BALB/c鼠拉颈处死，75％酒精全身浸泡，超净台内，无菌操作下用剪刀剪开腹部皮肤，暴露腹膜，用注射器腹腔注入RPMI 1640基础培养液5mL，反复冲洗，回收冲洗液，1000rpm，离心5分钟，留沉淀，用RPMI 1640筛选培养液(含HAT的RPMI 1640完全培养液中)重悬，调整细胞浓度1×105个/mL，加入96孔板，150μL/孔，37℃，5％CO2培养过夜。
(2)免疫脾细胞的制备
小鼠末次免疫后三天，在无菌条件下取出脾脏，置于平皿中，RPMI 1640基础培养液冲洗一次，放于小烧杯的尼龙网上磨碎过滤，制成细胞悬液。离心，弃上清，RPMI 1640基础培养液重悬，如此重复三次，计数。
(3)骨髓瘤细胞的制备
小鼠骨髓瘤细胞Sp2/0(菲鹏生物股份有限公司保存)经8-氮鸟嘌呤筛选后，培养至对数生长期，取两大瓶制成细胞悬液，离心，弃上清，用RPMI1640基础培养液重悬，如些重复三次，计数。
(4)细胞融合及HAT选择杂交瘤
将骨髓瘤细胞与免疫脾细胞按1∶10比例混合，在50mL塑胶离心管内用 RPMI 1640基础培养液洗1次，1200rpm，离心8分钟。弃上清，将细胞混匀，缓慢加入1mL 50％的PEG1500融合，融合1分钟后加入15mL的RPMI1640基础培养液终止细胞融合。1000rpm，离心5分钟。弃上清，用50mL的RPMI 1640筛选培养液轻轻混悬，平分于10块铺有饲养细胞的96孔板，50μL/孔，37℃，5％CO2培养。培养至第六天，换HT培养液(含HT的RPMI1640完全培养液)两次。
(5)抗体的检测
用0.06M pH9.6碳酸缓冲溶液稀释PK2-NGAL蛋白使其终浓度为2μg/mL。每孔0.1mL加入96孔聚苯乙烯板，37℃孵育2小时或4℃过夜。次日，用含10％小牛血清或1％脱脂奶粉的0.02M pH7.2PBS，0.15mL/孔，37℃封闭2小时，用于检测。上述杂交瘤细胞重组融合后第七天，取细胞上清稀释不同倍数后取0.1mL于上述96孔检测板中，37℃30分钟，PBST洗五次后加入2000倍稀释的辣根过氧化酶标记的羊抗鼠IgG(菲鹏生物股份有限公司生产，货号GRCGAMS001)，37℃30分钟同上洗后，每孔加入100μL含0.1％(M/V)邻苯二胺，0.1％(V/V)双氧水，pH5.0柠檬酸磷酸缓冲液，37℃15分钟，加入稀硫酸溶液，每孔50μL，测450nm吸收值。RPMI 1640完全培养液作为阴性对照，共检测有杂交瘤细胞的384孔，最终获得32株稳定分泌抗人NGAL的细胞株。细胞培养上清效价2.28×103以上。
3.单克隆抗体的制备
选6-8周健壮的BALB/c小鼠，每只小鼠腹腔注射0.5mL的降植烷；10天后腹腔注射1×106个杂交瘤细胞。接种细胞7～10天后可产生腹水，密切观察动物的健康状况与腹水征象，待腹水尽可能多，而小鼠频于死亡之前，处死小鼠，用滴管将腹水吸入试管中，一般一只小鼠可获5～10mL腹水。收集腹水，离心取上清，用3倍体积的PBS稀释后滤纸过滤。将所得的滤液在1mL/min的流速下加到一个已用PBS平衡的蛋白G亲和层析柱(GE公司)。然后用PBS以1mL/min的流速洗涤未被蛋白G吸附的物质直至在OD280nm下的吸附值达到基线为止。再用0.1M的甘氨酸洗脱液(pH2.5)洗脱并回收 该抗体。所回收的抗体立即用0.1M Tris(pH8.8)中和，跑胶测浓度。上述可分泌抗人NGAL杂交瘤腹水抗体效价2.77×106以上。
4.单克隆抗体表位鉴定
用0.06M pH9.6碳酸缓冲溶液稀释纯化好的待鉴定单抗使其终浓度为1μg/mL。每孔0.1mL加入96孔聚苯乙烯板，37℃孵育2小时或4℃过夜。次日，用含10％小牛血清或1％脱脂奶粉的0.02M pH7.2PBS，0.15mL/孔，37℃封闭2小时，加入2000倍稀释的辣根过氧化酶标记的NGAL表位鉴定抗原，37℃30分钟，PBST洗5次，拍干，每孔加入100μL含0.1％(M/V)邻苯二胺，0.1％(V/V)双氧水，pH5.0柠檬酸磷酸缓冲液，37℃15分钟，加入稀硫酸溶液，每孔50μL，测450nm吸收值，根据反应区分表位。
实施例3、NGAL检测试剂盒的制备及调试
1.缓冲液的配制
缓冲液为25mM Tris+25mM甘氨酸+0.1％BSA+50mMNaCl+0.5％吐温20+0.1％NaN3+25mMMgCl2+0.1％EDTA，pH8.2；
2.检测液的配制
采用化学交联法进行抗体和乳胶的连接，不同表位分别取一株单抗平均混合。经调试杂交瘤细胞株NGAL-3B5、杂交瘤细胞株NGAL-2D8和杂交瘤细胞株NGAL-4F6三株杂交瘤细胞分泌的单抗按照质量比为1∶1∶1平均混合后产品性能较好，步骤如下：
1)活化：20mg微球+1.4mL缓冲液+2mgEDC+6mgNHS室温震荡反应15min，20000rpm离心30min；
2)偶联：弃上清加入1mL 5mM CB超声，再加入混合后抗体(40μgAb/mgLatex)37度偶联1h后离心；
3)封闭：加入等量的0.5M甘氨酸+2％BSA，pH8.237度封闭1h或4度封闭过夜，20000rpm离心30min；
4)清洗：25mM Tris+50mM甘氨酸+0.1％NaN3，pH8.2悬浮，清洗， 20000rpm离心30min
5)保存：25mM Tris+50mM甘氨酸+0.1％BSA+0.1％NaN3+0.2％吐温+100mM NaCl+0.1％EDTA，pH8.2悬浮；
6)检测：
本发明用迈瑞BS-480采用终点法检测，波长570nm，时间点52-5482，R2∶Sample＝150μL∶100μL∶4μL
3.工作曲线的制定
采用标准品(重组NGAL)，选择7点校准，重组NGAL含量分别为0、178、356、712、1424、2848、5696ng/mL，按照上述测定步骤得到的NGAL的校准曲线(如图2所示)。图2中曲线上每个点代表一个含量的标准品，其中X轴表示NGAL的含量，y轴表示吸光度。
4.灵敏度测定
检测20次水和10ng/mL低值样本，记录吸光度数值，见表1。
表1：灵敏度实验数据
Figure PCTCN2016112283-appb-000001
Figure PCTCN2016112283-appb-000002
根据表1，计算平均值和标准偏差，算出最低检出限(LLD)＝0.2+3*1.046297＝3.34ng/mL，试剂盒灵敏度＝10*3.34/34.83＝0.9589ng/mL。
5.临床相关性
使用本发明的NGAL试剂盒和对照试剂盒(某国际知名公司NGAL试剂盒)，采用迈瑞BS-480全自动生化分析仪对50份人血清进行测定，对测定值进行相关性分析，测定结果见图3。
由图3可以看出，结果显示本发明的NGAL试剂盒与对照试剂盒的相关性很高。
6.NGAL试剂盒的稳定性
对NGAL试剂盒进行37度3天、7天、10天热破实验，数据结果见表2和表3。
表2：稳定性实验数据
Figure PCTCN2016112283-appb-000003
Figure PCTCN2016112283-appb-000004
表3：稳定性波动数据
Figure PCTCN2016112283-appb-000005
从表3数据可以看出，测试波动均在10％以内，说明本发明的NGAL试剂盒的稳定性合格。
以上所述实施例仅表达了本发明的一种或几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，其特征在于，包括：
    检测液，所述检测液中含有偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒；及
    标准品，所述标准品为具有天然构型的重组中性粒细胞明胶酶相关脂质运载蛋白。
  2. 根据权利要求1所述的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，其特征在于，所述偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒的粒径为80nm～220nm。
  3. 根据权利要求1所述的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，其特征在于，所述检测液中，所述偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒的浓度为1g/L～5g/L。
  4. 根据权利要求1所述的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，其特征在于，所述偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒中，抗人中性粒细胞明胶酶相关脂质运载蛋白抗体和胶乳颗粒的质量比为10～200∶1000。
  5. 根据权利要求4所述的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，其特征在于，所述
    胶乳颗粒的材料为羧基化的聚苯乙烯胶乳或者是氨基化的聚苯乙烯胶乳。
  6. 根据权利要求4所述的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，其特征在于，所述抗人中性粒细胞明胶酶相关脂质运载蛋白抗体为至少两种鼠抗人单克隆抗体形成的混合物。
  7. 根据权利要求4或6所述的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，其特征在于，所述抗人中性粒细胞明胶酶相关脂质运载蛋白抗体选自单克隆抗体NGAL-3B5、单克隆抗体NGAL-2D8和单克隆抗体NGAL-4F6 中的至少两种；
    所述单克隆抗体NGAL-3B5为杂交瘤细胞株NGAL-3B5分泌得到，所述杂交瘤细胞株NGAL-3B5的保藏号为CCTCC NO：C2016214；
    所述单克隆抗体NGAL-2D8为杂交瘤细胞株NGAL-2D8分泌得到，所述杂交瘤细胞株NGAL-2D8的保藏号为CCTCC NO：C2016215；
    所述单克隆抗体NGAL-4F6为杂交瘤细胞株NGAL-4F6分泌得到，所述杂交瘤细胞株NGAL-4F6的保藏号为CCTCC NO：C2016216。
  8. 根据权利要求1所述的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，其特征在于，所述偶联了抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的胶乳颗粒为抗人中性粒细胞明胶酶相关脂质运载蛋白抗体和胶乳颗粒通过化学交联法结合形成。
  9. 根据权利要求1所述的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，其特征在于，所述检测液还包括20mmol～100mmol的缓冲液、50mmol～500mmol无机盐、0.1wt％～5wt％的稳定剂和0.01wt％～0.2wt％防腐剂，所述检测液的pH为7.0～8.5；
    所述缓冲液为Tris缓冲液或磷酸盐缓冲，所述无机盐选自氯化钠、氯化钾、氯化镁和硫酸钾中的至少一种，所述稳定剂选自小牛白蛋白、明胶、甘氨酸、蔗糖、吐温20和TritonX-100中的至少一种，所述防腐剂选自叠氮钠和硫柳汞中的至少一种。
  10. 根据权利要求1所述的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒，其特征在于，所述具有天然构型的重组中性粒细胞明胶酶相关脂质运载蛋白为：
    (a)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸编码得到的多肽；
    (b)、与SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或者
    (c)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸，其中一个或 多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。
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