WO2020200315A1 - 经修饰的苯丙氨酸氧化酶酶原及其用途 - Google Patents

经修饰的苯丙氨酸氧化酶酶原及其用途 Download PDF

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WO2020200315A1
WO2020200315A1 PCT/CN2020/083317 CN2020083317W WO2020200315A1 WO 2020200315 A1 WO2020200315 A1 WO 2020200315A1 CN 2020083317 W CN2020083317 W CN 2020083317W WO 2020200315 A1 WO2020200315 A1 WO 2020200315A1
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seq
sequence
caspase
coagulation factor
zymogen
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French (fr)
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王辰
肖飞
张俊华
许小毛
邹丽辉
李传保
王萌
苏斐
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北京医院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

  • the application relates to a modified zymogen and its use, belonging to the field of protease activity detection.
  • proteases there are many kinds of proteases in the human body, but there is no direct, specific, sensitive, fast and effective method that can be widely used to detect the activities of various proteases.
  • the factor IIa in the system is also called thrombin, which belongs to the serine protease, which is the key link of thrombosis and hemostasis in the blood coagulation system.
  • the thrombin in the circulating blood exists in the form of inactive prothrombin, and the normal human body contains only a very small amount of active thrombin.
  • thrombin detection methods are roughly divided into indirect detection methods, including the determination of prothrombin fragment 1+2 (F1+2), fibrinopeptide A, soluble fibrin monomer complex, and thrombin-antithrombin complex Direct detection method, including chromogenic substrate method and fluorescence method.
  • the fibrinolytic system in the body is closely related to the coagulation system. Plasminogen in the fibrinolytic system is activated by streptokinase or urokinase and becomes plasmin (Plasmin), which can specifically degrade fibrin gel. It is broken down into soluble products, and the level of fibrinogen in the body is reduced through negative feedback effects, thereby avoiding excessive aggregation of fibrin. At present, there is no report about directly detecting the plasmin activity in the physiological state and activated state in the blood.
  • the coagulation test results reflect the overall performance of the coagulation system, and it is difficult to locate a certain coagulation factor.
  • the existing ELISA method for detecting coagulation factors only detects the presence and content of a specific protein, but does not reflect the activity of the enzyme.
  • the antibody titer and specificity issues in this method limit the standardization of test results.
  • the present invention provides a modified phenylalanine oxidase proenzyme (the proenzyme of L-phenylalanine oxidase, abbreviated as proPAO), characterized in that: the modification is in the proenzyme of L-phenylalanine oxidase
  • proPAO the proenzyme of L-phenylalanine oxidase
  • a specific recognition site sequence for protease is introduced between the leader sequence and the ⁇ subunit and between the ⁇ subunit and the ⁇ subunit in the sequence.
  • the phenylalanine oxidase proenzyme is derived from bacteria, such as Pseudomonas Sp.P-501, Pseudomonas donghuensis, Ralstonia solanacearum UW551 or Onion Burkholderia cepacia ATCC 25416.
  • the protease is selected from the coagulation cascade protease, such as coagulation factor IIa, Va, Vila, Villa, IXa, Xa, XIa, or XIIa; plasmin; cysteine-containing aspartic protease, such as Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10 or Caspase-11; complement pathway proteases, such as factors C1, C2, C3, C4, C5, C6, C7, C8, C9 or C3/C5 conversion enzyme; MMP family protease; NisP enzyme.
  • the coagulation cascade protease such as coagulation factor IIa, Va, Vila, Villa, IXa, Xa, XIa, or XIIa
  • plasmin cysteine-containing aspartic protease,
  • the specific recognition site sequence of the protease is shown in any one of SEQ ID NO: 8-44, 67, for example, shown in any one of SEQ ID NO: 9-12.
  • the cleavage sequence of factor IIa includes but is not limited to Xaa Xaa Xab Arg Xac (SEQ ID NO: 8), and the cleavage site is between Arg and Xac, where "Xaa” is any amino acid residue, and "Xab” is preferred Amino acid residues from Pro, Ala, Gly or Val, "Xac” is an amino acid residue preferably from Ser, Ala, and Gly, preferably Leu Val ProArg Gly (SEQ ID NO: 10); the cleavage sequence of factor IIa is also It can be Leu Arg Pro Arg (SEQ ID NO: 9) (the cleavage site is after Arg), Phe Pro Arg (SEQ ID NO: 11) (the cleavage site is after Arg) and Gly Arg Gly (SEQ ID NO: 12) ) (The cutting site is between Arg and Gly) and so on.
  • the cleavage sequence of factor Vila includes but is not limited to Leu Ile Gln Arg (SEQ ID NO: 13).
  • the cleavage sequence of coagulation factor IXa includes but is not limited to Xaa Xaa Gly Arg (SEQ ID NO: 14), "Xaa” is any amino acid residue, preferably Pro Gln Gly Arg (SEQ ID NO: 15).
  • the cleavage sequence of factor Xa includes but is not limited to Xad Xaa Xae Xaf (SEQ ID NO: 16), where "Xaa” is any amino acid residue, “Xad” is an amino acid residue preferably selected from Ala or Ile, and “Xae” is Preferably amino acid residues from Pro, Phe or Gly, “Xaf” is an amino acid residue selected from Arg or Lys, preferably Ile Glu Gly Arg (SEQ ID NO: 17) and Ile Asp Gly Arg (SEQ ID NO: 18) Wait.
  • the cleavage sequence of coagulation factor XIa includes but is not limited to Xag Xah Thr Arg (SEQ ID NO: 19), where "Xag” is an amino acid residue preferably from Lys or Asp, and "Xah” is an amino acid residue preferably from Phe or Leu , Preferably Lys Leu Thr Arg (SEQ ID NO: 20).
  • the cleavage sequence of coagulation factor XIIa includes but is not limited to Thr Ser Thr Arg (SEQ ID NO: 21).
  • the cleavage sequence of Plasmin includes but is not limited to Xaa Xaa Xai Xaj (SEQ ID NO: 22), and the cleavage site is between Xai and Xaj, where "Xaa” is any amino acid residue, and "Xai” is preferably from Arg Or the amino acid residue of Lys, "Xaj” is an amino acid residue preferably selected from Ala, Ser, Gly or Arg, preferably Gly Tyr Arg Ala (SEQ ID NO: 23) and Pro Ala Lys Ala (SEQ ID NO: 24).
  • the cleavage sequence of Caspase-2 includes but is not limited to Asp Glu Xaa Asp (SEQ ID NO: 25), where "Xaa” is any amino acid residue.
  • the cleavage sequence of Caspase-3 includes but is not limited to Xak Glu Val Asp (SEQ ID NO: 26), wherein "Xak” is an amino acid residue preferably from Asp or Glu, preferably Asp Glu Val Asp (SEQ ID NO: 27).
  • the cleavage sequence of Caspase-6 includes but is not limited to Xaa Glu Xaa Asp (SEQ ID NO: 28), where "Xaa” is any amino acid residue.
  • the cleavage sequence of Caspase-7 includes but is not limited to Asp Xaa Xaa Asp (SEQ ID NO: 29), where "Xaa” is any amino acid residue.
  • the cleavage sequence of Caspase-8 includes but is not limited to Xal Xam Thr Asp (SEQ ID NO: 30), wherein "Xal” is an amino acid residue preferably derived from Asp or Leu, and "Xam” is an amino acid residue preferably derived from Glu or Ser , Preferably Asp Glu Thr Asp (SEQ ID NO: 31).
  • the cleavage sequence of Caspase-9 includes but is not limited to Xaa Xan Xaa Asp (SEQ ID NO: 32), wherein "Xaa” is any amino acid residue, and “Xan” is an amino acid residue preferably selected from Asp or Glu.
  • the cleavage sequence of Caspase-10 includes but is not limited to Xaa Xao Xap Asp (SEQ ID NO: 33), wherein "Xaa” is any amino acid residue, and “Xao” is an amino acid residue preferably selected from Glu, Gln or Ser, " Xap” is an amino acid residue preferably selected from Thr or Val.
  • the cleavage sequence of Caspase-14 includes but is not limited to Leu Glu Xaa Asp (SEQ ID NO: 34), where "Xaa” is any amino acid residue.
  • the cleavage sequence of the complement pathway C3/C5 convertase includes but is not limited to Gln Leu Gly Arg Leu His Met Lys (SEQ ID NO: 35) (the cleavage site is between Arg and Leu) and Gly Leu Ala Arg Ser Asn Leu Asp( SEQ ID NO: 36) (The cleavage site is between Arg and Ser).
  • the cleavage sequence of MMP-8 includes but is not limited to Gly Xaq Xaa Gly (SEQ ID NO: 37), wherein "Xaa” is any amino acid residue, and "Xaq” is an amino acid residue preferably selected from Pro, Ala or Ser.
  • the cleavage sequence of MMP-11 includes but is not limited to Xaa Ala Ala Ala (SEQ ID NO: 38), where "Xaa” is any amino acid residue.
  • the cleavage sequence of MMP-12 includes but is not limited to Gly Xar Xas Xas (SEQ ID NO: 39), where "Xar” is an amino acid residue preferably selected from Pro, Ala or Gly, and "Xas” is an amino acid preferably selected from Ala or Gly Residues.
  • the cleavage sequence of MMP-13 includes but is not limited to Gly Pro Xaa Gly Xat (SEQ ID NO: 40) (the cleavage site is between Gly and Xat), where "Xaa” is any amino acid residue, and "Xat” is preferably from The amino acid residue of Leu, Ile or Val is preferably Gly Pro Ala Gly Leu (SEQ ID NO: 41).
  • the cleavage sequence of MMP-20 includes but is not limited to Pro Xaa Leu Pro Xau (SEQ ID NO: 42) (the cleavage site is between Pro and Xau), where "Xaa” is any amino acid residue, and "Xau” is preferably from The amino acid residue of Leu or Met is preferably Pro Ala Leu Pro Leu (SEQ ID NO: 43) or Pro Ala Leu Pro Met (SEQ ID NO: 44).
  • the zymogen sequence of the phenylalanine oxidase is shown in SEQ ID NO:1.
  • the modified phenylalanine oxidase proenzyme sequence is as described in any one of SEQ ID NOs: 2, 4-7.
  • the present invention provides the use of the modified phenylalanine oxidase zymogen of the first aspect for detecting protease activity in a sample.
  • the present invention also provides a method for detecting protease activity in a sample, the method comprising contacting the modified phenylalanine oxidase zymogen of the first aspect with the sample.
  • the sample may be body fluid, whole blood, plasma, serum or tissue.
  • the phenylalanine oxidase proenzyme derived from Pseudomonas Sp.P-501 is used as the reaction substrate, and the specific recognition sequence of coagulation factor IIa is introduced into it through genetic engineering technology, and phenylalanine is used.
  • the redox and color reaction of acid oxidase (PAO) and the substrate are used to construct the time curve between the concentration of coagulation factor IIa and the absorbance of the reaction product, and the direct detection system of coagulation factor IIa activity is established.
  • clinical samples were used to verify the feasibility of the detection system, confirming that the detection method is suitable for the detection of sodium citrate anticoagulated plasma samples.
  • the present invention constructs a complete detection method for coagulation factor IIa, and through the two-stage cascade reaction of coagulation factor IIa and oxidoreductase, the detection range is widened and the detection sensitivity is improved.
  • the use of this detection system requires an additional step of zymogen activation cascade to amplify the concentration signal of factor Xa.
  • the intermediate protease meets the basic amplification requirements, that is, no Exist in the human body, the recognition sequence is highly specific, the reaction pH is neutral, and the absorbance change of the plasma sample can be detected within 30 minutes.
  • the intermediate protease is a modified NisP protease protease (proNisP) that includes an enzyme-specific recognition site sequence between the leader peptide and the catalytic subunit.
  • proNisP modified NisP protease protease
  • the modified proNisP is derived from Streptococcus lactis.
  • the use of streptokinase to pre-treat plasma can prevent the coagulation of plasma samples in the fully activated state.
  • plasminogen can be directly activated by streptokinase to generate plasmin, which causes an enzyme-linked activation reaction to establish a direct detection system for plasmin activity.
  • phenylalanine oxidase zymogen is used as the reaction substrate, and the specific recognition sequence of Caspase-3 is constructed through genetic engineering technology, and the redox and color reaction of PAO and the substrate are used.
  • the reaction substrate phenylalanine oxidase zymogen
  • the specific recognition sequence of Caspase-3 is constructed through genetic engineering technology, and the redox and color reaction of PAO and the substrate are used.
  • clinical samples are used to verify the feasibility of the detection system, confirming that the detection method is suitable for the detection of serum samples.
  • C5 converting enzyme enzyme activity In the detection of C5 converting enzyme enzyme activity, using phenylalanine oxidase zymogen as the reaction substrate, the specific recognition sequence of C5 converting enzyme was constructed through genetic engineering technology, and the redox and color reaction of PAO and the substrate were used. Establish a direct detection system for C5 converting enzyme activity in the complement system. At the same time, clinical samples are used to verify the feasibility of the detection system, confirming that the detection method is suitable for the detection of serum samples.
  • the plasma sample used is sodium citrate anticoagulated plasma sample or heparin anticoagulated plasma sample.
  • pretreatment of the sample with streptokinase or urokinase is also included.
  • the redox and color reaction of the activated phenylalanine oxidase and the substrate are included.
  • the present invention provides an isolated nucleic acid encoding the modified phenylalanine oxidase zymogen described in the first aspect.
  • the present invention provides an expression vector comprising the isolated nucleic acid of the third aspect.
  • the present invention provides a host cell transformed or transfected with the expression vector of the fourth aspect.
  • the present invention provides a kit for detecting protease activity in a sample, which includes the modified phenylalanine oxidase zymogen described in the first aspect.
  • the kit further includes a modified proNisP, which includes an enzyme-specific recognition site sequence between the leader peptide and the catalytic subunit, such as a specific recognition sequence for coagulation factor Xa.
  • a modified proNisP which includes an enzyme-specific recognition site sequence between the leader peptide and the catalytic subunit, such as a specific recognition sequence for coagulation factor Xa.
  • the inactive L-phenylalanine oxidase zymogen (proPAO) from Psuedomonas Sp.P-501 is basically composed of a leader sequence of 14 amino acid residues, an ⁇ subunit of 92 amino acid residues, and a dipeptide ( Ile-Lys) and a ⁇ subunit of 605 residues.
  • proPAO can be hydrolyzed by the protease Pronase and Trypsin into ⁇ subunit and ⁇ subunit fragments lacking the first lysine residue.
  • the activity of the hydrolyzate is similar to that containing ⁇ .
  • PAO activity of the activated form of subunit and ⁇ subunit fragment is equivalent (Suzuki, H. et al. Sequencing and expression of the L-phenylalanine oxidase gene from Pseudomonas sp. P-501. Proteolytic activation of the proenzyme.
  • the ⁇ -subunit referred to in this article includes the form that lacks a lysine at the N-terminus, and the so-called leader sequence can correspond to an additional lysine at the C-terminus.
  • Phenylalanine oxidase refers to phenylalanine 2-monooxygenase (EC 1.13.12.9), which mainly catalyzes the production of 2-phenylacetamide or 3-phenylpyruvate from L-phenylalanine.
  • the substance also includes ⁇ -2-thiophene alanine, L-tyrosine, L-methionine and so on.
  • Proenzyme A precursor form of an enzyme with a specific activity of an enzyme, so it is generally called an inactive form of enzyme.
  • the zymogen referred to herein can refer to the precursor form of the enzyme that is activated by enzyme digestion in the detection method.
  • the zymogen can be activated to the corresponding active enzyme by other enzymes that cause the activation cascade.
  • Modification mutation (replacement), insertion (addition) or deletion of one or more amino acid residues in a polypeptide (such as an enzyme) to obtain a modified polypeptide.
  • a polypeptide such as an enzyme
  • Recognition site sequence refers to the amino acid sequence specifically recognized and cleaved by the enzyme in the detection.
  • recognition site sequence refers to the amino acid sequence specifically recognized and cleaved by the enzyme in the detection.
  • recognition site sequence refers to the amino acid sequence specifically recognized and cleaved by the enzyme in the detection.
  • recognition site sequence refers to the amino acid sequence specifically recognized and cleaved by the enzyme in the detection.
  • recognition site sequence refers to the amino acid sequence specifically recognized and cleaved by the enzyme in the detection.
  • protease precursor form that can be activated into active protease.
  • the proteases referred to herein include serine proteases, threonine proteases, cysteine proteases, aspartic proteases, metalloproteases or glutamic acid proteases, and any other enzymes that can cleave protein sequences.
  • Amino acid residues including but not limited to the amino acid residues consisting of the following amino acids: alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn , N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), group Acid (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and Valine (Val, V).
  • alanine three letter code: Ala, one letter code: A
  • arginine Arg,
  • Coding sequence The boundary of the coding sequence is generally determined by an open reading frame, which starts with a start codon (such as ATG, GTG or TTG) and ends with a stop codon (such as TAA, TAG or TGA).
  • the coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • Transformation refers to the introduction of DNA into a recipient host cell, which changes the genotype and subsequently leads to changes in the recipient cell.
  • Host cell refers to a cell transformed with a vector constructed using recombinant DNA technology and encoding at least one heterologous gene.
  • the preparation method of coagulation factor IIa specific substrate includes strain, plasmid, enzyme and culture medium, PCR amplification and construction of recombinant plasmid, induced expression of gene and purification of protein. Specific steps are as follows:
  • Escherichia coli DH5 ⁇ , BL21(DE3), cloning and expression plasmid vector pRSFDuet-1 was purchased from Novagen; DNA high-fidelity polymerase mixture was purchased from Tsingke; restriction endonuclease and DNA ligase were purchased from NEB Company; E. coli LB medium: Each liter contains Tryptone 10g, Yeast extract 5g, NaCl 10g, and the kanamycin concentration is 50mg/L.
  • proPAO i.e. SEQ ID NO:1
  • SEQ ID NO:1 The gene sequence encoding proPAO (i.e. SEQ ID NO:1) was synthesized for the whole gene, and three pairs of primers were designed, and the proPAO activation site (i.e. between the leader sequence and the ⁇ subunit, and the ⁇ subunit and the Between ⁇ subunits) two specific recognition sequences for coagulation factor IIa (amino acid sequence LRPR, SEQ ID NO: 9) are added. After double digestion with restriction enzymes, it was ligated to the expression vector pRSFDuet-1 digested with the same endonuclease, and the ligation product was transformed into E. coli DH5 ⁇ . The recombinant plasmid was screened and verified by double digestion and sequencing.
  • the primers for the above overlap extension PCR are as follows:
  • the underlined part is the modified proPAO upstream primer NcoI restriction site and downstream primer NdeI restriction site. Two restriction enzymes, NcoI and NdeI, are used.
  • the recombinant plasmid was transformed into E. coli BL21 (DE3). IPTG was added to the culture to induce expression. The induced protein was broken by ultrasound, centrifuged, and the supernatant was taken for nickel column affinity chromatography (product of QIAGEN) and desalting chromatography column (product of Amersham) to obtain purified coagulation factor IIa substrate-specific proPAO.
  • the reaction process can be divided into two steps:
  • PAO catalyzes the oxidative deamination reaction of the substrate L-phenylalanine (L-Phe) to generate ⁇ -phenylpyruvate, ammonia (NH 3 ) and hydrogen peroxide (H 2 O 2 ) .
  • L-Phe L-phenylalanine
  • NH 3 ammonia
  • H 2 O 2 hydrogen peroxide
  • Trinder reaction under the catalysis of horseradish peroxidase (HRP), hydrogen peroxide (H 2 O 2 ) reacts with phenol derivatives (TOOS) and 4-aminoantipyrine (4-AAT) Quinones are produced, which are red-purple, with the maximum absorption peak at 550-560nm, and the color intensity is proportional to the concentration of hydrogen peroxide.
  • the signal detection of this system selects dual-wavelength spectrophotometry.
  • the wavelength of the maximum absorption peak is 550-560nm as the measuring wavelength, and the wavelength of the minimum absorption in the visible light range is 700nm as the combined wavelength. Abs 555nm -Abs 700nm was used to process the absorbance detection data as the detection analysis signal.
  • the enzyme reaction rate increases with the increase of the substrate proPAO concentration.
  • a high concentration of proPAO will cause the spatial distance of the substrate to be too close, resulting in a higher background reaction of proPAO. Therefore, the proPAO concentration with a relatively high reaction rate is selected as the final concentration of the detection reaction.
  • the concentration of other substrates L-Phe, HRP, TOOS and 4-AAT
  • the pH value of the reaction were optimized to make the reaction system have the maximum absorbance change rate.
  • coagulation factor Xa is located upstream of coagulation factor IIa.
  • an intermediate zymogen is selected to increase a zymogen activation reaction to amplify the concentration signal of factor Xa.
  • NisP proenzyme Niin leader peptide-processing serine proenzyme, proNisP
  • the recombined proNisp gene sequence is synthesized from the whole gene, and the specific recognition sequence IEGR (SEQ ID NO: 17) of factor Xa is added between the leader peptide sequence and the catalytic subunit of the zymogen.
  • three pairs of primers were designed, and two NisP specific recognition sequences ASPRI (SEQ ID NO: 67) were added to the proPAO activation site by overlap extension PCR.
  • the primers for the above overlap extension PCR are as follows:
  • the underlined part is the modified proPAO upstream primer NcoI restriction site and downstream primer NdeI restriction site.
  • Two restriction enzymes, NcoI and NdeI, are used.
  • the constructed recombinant zymogen proNisp and proPAO were respectively connected to the expression vector pRSFDuet-1 and transformed into E. coli BL21 (DE3), respectively, to induce gene expression and protein purification.
  • the induced expression of proNisp and the purification of protein are as follows: IPTG is added to the culture to induce expression.
  • the induced expression of the protein is ultrasonically broken, centrifuged, and the supernatant is taken for nickel column affinity chromatography (product of QIAGEN) and desalting chromatography column (product of Amersham) to obtain protein products.
  • the pure commercial coagulation factor Xa product of NEB
  • the purified recombinant proNisP zymogen were added to the above recombinant proPAO, incubated at 37° C., and SDS-PAGE verified the hydrolysis efficiency.
  • Detection of coagulation factor Xa activity in clinical specimens use clinical sodium citrate anticoagulated plasma specimens, and supplement sufficient CaCl 2 and TF in the reaction system to fully activate coagulation factor Xa in the plasma to be tested.
  • the specimen is pre-treated with streptokinase to degrade the fibrin in it, and then the processed specimen is tested.
  • proPAO Pseudomonas Sp.P-501
  • proPAO Plasmin specific recognition sequences GYRA (SEQ ID NO: 23) were added to the activation site of the zymogen proPAO by overlap extension PCR.
  • GYRA Plasmin specific recognition sequences
  • the primers for the above overlap extension PCR are as follows:
  • the underlined part is the modified proPAO upstream primer NcoI restriction site and downstream primer NdeI restriction site.
  • Two restriction enzymes, NcoI and NdeI, are used.
  • the subsequent implementation content can refer to the coagulation factor IIa activity detection method. It should be noted that, unlike the coagulation factor IIa and Xa, in the embodiment of the plasmin activity detection method, streptokinase can directly activate plasminogen in plasma It generates plasmin, which causes an enzyme-linked activation reaction to establish a direct detection system for plasmin activity.
  • proPAO Pseudomonas Sp.P-501 gene as a template, design three pairs of primers, and add two Caspase-3 specific recognition sequences DEVD (SEQ ID NO: 27) at the activation site of zymogen proPAO by overlap extension PCR . After double digestion with restriction enzymes, it was ligated to the expression vector pRSFDuet-1 digested with the same endonuclease, and the ligation product was transformed into E. coli DH5 ⁇ . The recombinant plasmid was screened and verified by double digestion and sequencing.
  • the primers for the above overlap extension PCR are as follows:
  • the underlined part is the modified proPAO upstream primer NcoI restriction site and downstream primer NdeI restriction site.
  • Two restriction enzymes, NcoI and NdeI, are used.
  • Enzyme activity unit of Caspase-3 can hydrolyze acetyl-Asp-Glu-Val-Asp-p-nitroaniline (Ac-DEVD-pNA), release p-nitroaniline (pNA) part, can be at 405nm Determination.
  • the enzyme activity unit of Caspase-3 can be defined by detecting the cleavage activity of recombinant Caspase-3 on the chromogenic substrate Ac-DEVD-pNA.
  • the implementation content of the subsequent substrate proPAO activation reaction can refer to the coagulation factor IIa activity detection method. It should be noted that, unlike the detection method in the coagulation system, in all the implementations of the Caspase activity detection method, the clinical specimen used is serum ingredient. In addition, there is no need to use an activator in the reaction, and a direct detection system for Caspase-3 activity can be established by directly detecting the activation reaction of Caspase-3 in the serum to proPAO containing a specific cleavage sequence.
  • proPAO Pseudomonas Sp.P-501
  • proPAO Pseudomonas Sp.P-501
  • two C5 invertase specific recognition sequences QLGRLHMK SEQ ID NO: 35
  • the ligation product was transformed into E. coli DH5 ⁇ .
  • the recombinant plasmid was screened and verified by double digestion and sequencing.
  • the primers for the above overlap extension PCR are as follows:
  • the underlined part is the modified proPAO upstream primer NcoI restriction site and downstream primer NdeI restriction site.
  • Two restriction enzymes, NcoI and NdeI, are used.
  • the subsequent implementation content can refer to the Caspase-3 activity detection method, which can directly detect the activation reaction of the C5 convertase in the serum to the proPAO containing the specific cleavage sequence to establish a direct detection system for the C5 convertase activity in the complement system.
  • Figure 1 is the SDS-PAGE detection result of the purified product of the coagulation factor IIa specific substrate proPAO (containing the LRPR recognition sequence), where: M is the protein marker.
  • Figure 2 shows the SDS-PAGE detection result of the enzymatic hydrolysis of coagulation factor IIa on the specific substrate proPAO (containing the LRPR recognition sequence), where: M is the protein marker.
  • Figure 3 shows the quantitative detection results of the enzymatic hydrolysis activity of the pure coagulation factor IIa on the factor IIa specific substrate proPAO (containing the LRPR recognition sequence).
  • Figure 4 shows the linear relationship between coagulation factor IIa activity units and the corresponding reaction rate equation parameters.
  • Figure 5 shows the detection result of the enzymatic hydrolysis activity of the coagulation factor IIa in the plasma against the substrate proPAO (containing the LRPR recognition sequence) after pretreatment with streptokinase.
  • Figure 6 shows the SDS-PAGE detection results of the purified products of proNisP (containing the IEGR recognition sequence) and NisP specific substrate proPAO (containing the ASPRI recognition sequence) containing two coagulation factor Xa cleavage sites, where: 20 and 250 are nickel, respectively
  • Figure 7 shows the product NisP after the coagulation factor Xa enzymatically hydrolyzes the factor Xa specific substrate proNisP (containing the IEGR recognition sequence), and the SDS-PAGE detection result of the product NisP on the NisP specific substrate proPAO (containing the ASPRI recognition sequence).
  • M is a protein marker.
  • Figure 8 shows the results of the enzyme-linked hydrolysis activity of coagulation factor Xa in plasma after pretreatment with streptokinase by enzymolysis of factor Xa specific substrate proNisP (containing IEGR recognition sequence) to NisP specific substrate proPAO (containing ASPRI recognition sequence).
  • Figure 9 shows the results of the enzymatic hydrolysis activity of plasma plasmin on the specific substrate proPAO (containing the GYRA recognition sequence) after activation of plasminogen by streptokinase.
  • Figure 10 shows the detection result of the cleavage activity of recombinant Caspase-3 on the synthetic substrate Ac-DEVD-pNA.
  • Figure 11 shows the detection results of the enzymatic activity of Caspase-3 on the specific substrate proPAO (containing the DEVD recognition sequence) in the serum of different subjects.
  • Sample 1 is a patient with small cell lung cancer and liver metastasis
  • Sample 2 is a patient with cardia disease
  • Sample 3 is a patient with suspected lymphoma
  • Sample 4 is a medical examiner.
  • Figure 12 shows the detection results of the enzymatic activity of C5Convertase on the specific substrate proPAO (containing the QLGRLHMK recognition sequence) in the serum of different subjects.
  • Sample 1 and Sample 2 are patients with rheumatic immune diseases
  • Sample 3 and Sample 4 are those undergoing physical examination.
  • a and B are the detection results of specific substrates proCPO (signal crayfish Pacifastacus leniusculus) and proDPO (Drosophila melanogaster) activated by coagulation factors, respectively.
  • Figure 14 shows the detection result of the enzymatic activity of the coagulation factor IIa in the plasma against the substrate proPAO (containing an LRPR recognition sequence) after pretreatment with streptokinase.
  • Figure 15 shows the enzymatic activity detection result of coagulation factor IIa in plasma after pretreatment with streptokinase against Staphylococcus aureus-derived SplB protease specific substrate proPAO (containing WELQ, SEQ ID NO: 68 recognition sequence) .
  • Fig. 16 is a flow chart of the zymogen activation reaction of proPAO modified with specific protease recognition sequence to determine the corresponding protease activity.
  • protease activity detection include coagulation cascade protease (including multiple coagulation factors), plasmin, cysteine-containing aspartic protease (including Caspase family members), and complement pathway protease (including various complement components) ) And matrix metalloproteinases (including MMP family members).
  • coagulation cascade protease including multiple coagulation factors
  • plasmin including multiple coagulation factors
  • cysteine-containing aspartic protease including Caspase family members
  • complement pathway protease including various complement components
  • matrix metalloproteinases including MMP family members
  • PCR reaction system 1.1 ⁇ PCR mix buffer 20 ⁇ l, upstream and downstream primers (10 ⁇ M) each 1 ⁇ l, template 0.2 ⁇ l. Reaction conditions: thermal denaturation at 94°C for 5min; denaturation at 94°C for 45s, annealing at 60°C for 45s, extension at 72°C for 30s (when primers PAO A1 and PAO A2 are used) or 2 minutes (when primers PAO B1 and PAO B2 are used), a total of 30 cycles ; Extend at 72°C for 10 min.
  • the PCR products were electrophoresed in an agarose gel prepared by 0.5 ⁇ TAE, and the DNA recovery kit (Tiangen) was used to recover the target fragments. For the method, refer to its instructions. The recovered products were stored at -20°C for later use.
  • the A fragment amplified using primers PAO A1-45 and PAO A2-46 and the B fragment obtained using primers PAO B1-47 and PAO B2-48 are subjected to overlap extension PCR.
  • the primers used PAO A1-45 upstream and PAO B2-48 downstream.
  • PCR reaction system 1.1 ⁇ PCR mix buffer 20 ⁇ l, upstream and downstream primers (10 ⁇ M) each 1 ⁇ l, template A fragment and B fragment each 1 ⁇ l. Reaction conditions: thermal denaturation at 94°C for 5min; denaturation at 94°C for 45s, annealing at 60°C for 45s, extension at 72°C for 2min 10s, a total of 30 cycles; extension at 72°C for 10min.
  • the PCR product recovered by electrophoresis in agarose gel is the same as before.
  • a pure DNA fragment containing a coagulation factor IIa cleavage site (located between ⁇ subunit and ⁇ subunit) is obtained.
  • the recovered DNA fragments were digested with restriction enzymes, and the reaction system: 43 ⁇ l of recovered PCR product, 5 ⁇ l of 10 ⁇ restriction endonuclease buffer, 1 ⁇ l of NcoI (Thermal Scientific Company), and 1 ⁇ l of NdeI (Thermal Scientific Company). Mix well and enzymatically digest at 37°C overnight.
  • the digested PCR amplified gene was recovered by agarose electrophoresis, and it was connected with the plasmid vector pRSFDuet-1 recovered by the same two restriction enzyme digestion.
  • Ligation reaction system 12 ⁇ l of PCR amplified gene after digestion, 2 ⁇ l of plasmid vector after digestion, 2 ⁇ l of 10 ⁇ T4 ligase buffer, 0.2 ⁇ l of T4 ligase (Thermal Scientific), 3.8 ⁇ l of ddH 2 O. Mix well and react at 20°C for 2h.
  • Transformation of the ligation product Take 100 ⁇ l of competent E.coli DH5 ⁇ into a 1.5ml centrifuge tube, and add 10 ⁇ l of the ligation product. Place on ice for 30 minutes, heat shock at 42°C for 90 seconds, and place on ice for 2 minutes. Add 500 ⁇ l of LB medium and culture with shaking at 37°C for 45min to recover the bacteria.
  • Cultivation of transformed bacteria Centrifuge at 12,000 rpm for 1 min, discard the supernatant, resuspend the bacteria in 100 ⁇ l LB medium, and spread on a kanamycin-resistant LB plate. The plate was cultured in a 37°C incubator for 16h.
  • Recombinant plasmid identification pick a single colony into a reaction tube, add 10 ⁇ l ddH 2 O to resuspend and mix, take 1 ⁇ l as a template, and use primers PAO A1-45 and PAO B2-48 for colony PCR.
  • the PCR results were identified by agarose gel electrophoresis.
  • the monoclonal colonies with correct colony PCR were inoculated into 5ml kanamycin-resistant LB medium and cultured with shaking at 37°C for 12h.
  • a small amount of plasmid DNA was extracted using a plasmid extraction kit (Axygen company).
  • the primers used PAO A1-45 upstream and PAO B2-48 downstream.
  • the PCR reaction system and conditions are the same as (1).
  • a pure proPAO DNA fragment containing two coagulation factor IIa cutting sites is obtained, and the corresponding amino acid sequence is shown in SEQ ID NO: 2.
  • the aforementioned recombinant plasmid containing the modified proPAO DNA fragment with two coagulation factor IIa cleavage sites was transformed into E. coli BL21 (DE3). Pick a single colony on the plate, inoculate it into 5ml LB medium, and culture with shaking at 37°C for 12h. All the above cultures were inoculated into 400ml fresh LB medium and cultured with shaking at 37°C for 3h. When the OD 600nm of the bacterial solution reached 0.6, IPTG with a final concentration of 0.5mM was added to the culture, and the culture was continued at 20°C for 16h. The culture was collected in a 50ml centrifuge tube and centrifuged at 6000 rpm for 20 min at 4°C. Discard the supernatant.
  • Bacterial cell dissolution The cell pellet obtained by centrifugation is fully resuspended in buffer A [50mM KH 2 PO 4 /K 2 HPO 4 , 300 mM NaCl, pH 8.0].
  • Bacteria fragmentation choose ultrasonic fragmentation.
  • the ultrasonic breaking conditions are 20% power, ultrasonic 2s intermittent 4s, 5-30min. After the bacterial liquid was clear, centrifuged at 4°C, 13000 rpm for 30 min, and collected the supernatant.
  • Nickel column affinity chromatography add 0.8ml of 50% Ni-NTA suspension (QIAGEN company) to the supernatant, and mix at 4°C for 1 hour at a constant speed. The mixture is added to the chromatography column, the supernatant flows out under the action of gravity, and the Ni-NTA matrix precipitates in the chromatography column. Add 1 column volume of buffer A to the chromatography column, wash the chromatography column, and repeat three times. After buffer A has flowed out, add 5 ml buffer A containing 20 mM imidazole to the chromatography column to elute impurity proteins, and repeat three times.
  • elution buffer B [50mM KH 2 PO 4 /K 2 HPO 4 , 300 mM NaCl, 250 mM Imidazole, pH 8.0] to the chromatography column in portions to elute the target protein. Collect the protein eluate in a 1.5ml centrifuge tube.
  • Desalting column chromatography and storage Centrifuge the obtained target protein eluate at 4°C, 13000rpm for 30min, take the supernatant and use ATKA protein purifier (Amersham) for desalting chromatography to make the protein in buffer C [10mM KH 2 PO 4 /K 2 HPO 4 , pH 8.0], add glycerol to 10%, and store at -80°C for later use.
  • ATKA protein purifier Anamersham
  • glycerol add glycerol to 10%
  • a protein quantification kit (Beyotime) was used to determine the content of the purified product, and the target protein sample was subjected to SDS-PAGE electrophoresis to detect the protein expression and purification.
  • the electropherogram is shown in Figure 1.
  • Reagent R1 TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt);
  • Reagent R2 L-Phe, HRP, 4-AAT.
  • Absorbance detection reaction system pure blood coagulation factor IIa (different activity units), coagulation factor IIa substrate specific proPAO, reaction reagent R1, reaction reagent R2. Add the above components to the small wells of the microplate, use a microplate reader to shake and mix the reaction system, and detect the changes in absorbance at 555nm and 700nm over time.
  • ⁇ Abs Abs 555nm -Abs 700nm , with time t as the abscissa and ⁇ Abs as the ordinate, plot the scatter plots of the kinetics of FIIa (Sigma) pure enzyme dissolution in different activity units.
  • the test results are shown in Figure 3. It can be seen from Figure 3 that as the activity unit of coagulation factor IIa in the reaction system increases, the reaction rate at which the modified proPAO is cleaved increases.
  • Optimize the detection reaction conditions separately prepare detection reagents containing different substrate concentrations. Control other substrates and the reaction conditions unchanged, select reagents of different concentrations containing the same substrate, and use the specific substrate after hydrolysis of the pure coagulation factor IIa as the sample for reaction. Use a microplate reader to continuously monitor the absorbance change of the reaction system, and draw a scatter plot with time t as the abscissa and ⁇ Abs as the ordinate.
  • the determined optimal reaction substrate concentrations are 8mM TOOS, 7.5mM L-phenylalanine, 6mM 4-AAT, 5U/ml HRP and 0.20 ⁇ g/ ⁇ l modified proPAO.
  • the optimal reaction temperature is 37°C.
  • Optimizing the pH of the detection reaction under the optimal pH conditions for the activity of coagulation factor IIa and blood pH conditions, prepare a gradient pH phosphate buffer.
  • the specific substrate hydrolyzed by the pure product of coagulation factor IIa was used as the sample for reaction, and the absorbance change of the reaction system was continuously monitored by a microplate reader.
  • Use time t as the abscissa and ⁇ Abs as the ordinate to draw a scatter plot.
  • the optimal pH is 7.35-7.45.
  • Sample collection Select the general outpatient clinics of Beijing Hospital from June to September 2017 (non-cardiovascular disease, non-pregnancy, non-orthopedic inpatient review patients, non-thyroid disease and other patients with high risk of thrombosis, non-anticoagulation therapy and contraceptive use
  • the sodium citrate anticoagulated plasma sample of the patient), sodium citrate: blood 1:9 (the final concentration of sodium citrate is about 0.109mmol/L).
  • the plasma samples were mixed in groups of 10 to prepare a plasma plate. Take mixed plasma as a sample for testing.
  • Sample detection After adding the following components (mixed plasma sample, reaction reagent R1 (90 ⁇ l), reaction reagent R2 (90 ⁇ l) and specific substrate proPAO (10 ⁇ l)) into the microplate reader, set the reaction procedure of the microplate reader: shaking and mixing Detect the change of absorbance at 555nm and 700nm with time.
  • CaCl 2 is added to the detection reaction system of sodium citrate anticoagulated plasma specimens (plasma samples, R1, R2 and modified proPAO), and tissue factor (TF) is added at the same time to fully activate the coagulation factor IIa in the plasma to be tested.
  • Sample preparation Prepare a mixed plasma sample for sodium citrate anticoagulation, and prepare a pretreated plasma sample using 20 ⁇ l streptokinase + 100 ⁇ l mixed plasma sample.
  • the control sample was made with 20 ⁇ l ddH 2 O+100 ⁇ l mixed plasma specimen.
  • Add the following ingredients pretreatment sample/control sample (20 ⁇ l), reaction reagent R1 (90 ⁇ l), reaction reagent R2 (90 ⁇ l) and modified proPAO (10 ⁇ l)
  • pretreatment sample/control sample (20 ⁇ l), reaction reagent R1 (90 ⁇ l), reaction reagent R2 (90 ⁇ l) and modified proPAO (10 ⁇ l)
  • the test results are shown in Figure 5 (the substances in the box in the legend are premixed). It can be seen from Figure 5 that after pretreatment of plasma with streptokinase, activated coagulation factor IIa caused an enzyme-linked activation reaction.
  • Trinder reaction is a more mature common reaction in clinical biochemical tests.
  • the reaction conditions, substrate and peroxidase concentration are all based on the peroxidase reaction parameters.
  • the concentration of the remaining substrates and enzymes in the reaction system is much higher than the K m value.
  • the quantitative relationship between the absorbance value of signal detection and the concentration of coagulation factor IIa of the sample to be tested is analyzed.
  • the primary reaction stage there is a linear relationship between the absorbance value of signal detection and the concentration of coagulation factor IIa of the sample to be tested.
  • a modified proNisP (Streptococcus lactis) zymogen DNA fragment was synthesized first, and the leading peptide sequence and the catalytic subunit sequence of the DNA fragment contained blood coagulation.
  • the ligation product is transformed and the recombinant plasmid with the correct sequence is identified, and the recombinant plasmid is transformed into E. coli BL21 (DE3).
  • modified protein proNisP nickel column affinity chromatography
  • desalting chromatography For the steps of expression and purification, please refer to the expression and purification of coagulation factor IIa substrate-specific proPAO.
  • the target protein sample was subjected to SDS-PAGE electrophoresis to detect the expression and purification of the protein. The electrophoresis is shown in Figure 6.
  • Enzymatic hydrolysis verification of coagulation factor Xa Take pure bovine coagulation factor Xa (NEB company) and press the reaction system (pure factor Xa 0.5 ⁇ g, modified proNisP 2 ⁇ g, modified proPAO 6 ⁇ g, 30mM KH 2 PO 4 /Na 2 HPO 4 , pH 7.37), after incubating at 37°C for 2.5 hours, SDS-PAGE verified the hydrolysis efficiency.
  • the electropherogram is shown in Figure 7. It can be seen from Figure 7 that coagulation factor Xa can successfully cleavage modified proPAO by cleaving modified proNisP.
  • Each lane from left to right is a protein marker, uncut modified proNisP, coagulation factor Xa cleaves modified proNisP, coagulation factor Xa cleaves modified proPAO by cleaving modified proNisP, and uncut modified proPAO is not Cleaved modified proNisP and modified proPAO, protein marker.
  • the reaction system contains pure factor Xa or sodium citrate anticoagulated plasma sample (20 ⁇ l), modified proNisP (6 ⁇ l), modified proPAO (20 ⁇ l), Reagent R1 (90 ⁇ l), Reagent R2 (90 ⁇ l).
  • the reaction system contains pure factor Xa or sodium citrate anticoagulated plasma sample (20 ⁇ l), modified proNisP (6 ⁇ l), modified proPAO (20 ⁇ l), Reagent R1 (90 ⁇ l), Reagent R2 (90 ⁇ l).
  • Add the above components to the small wells of the microplate use a microplate reader to shake and mix the reaction system, and detect the changes in absorbance at 555nm and 700nm over time.
  • ⁇ Abs Abs 555nm -Abs 700nm , with time t as the abscissa and ⁇ Abs as the ordinate, draw the scatter plot of the enzymatic hydrolysis kinetics, and perform function fitting on each scatter plot to describe the corresponding The reaction rate.
  • coagulation factor IIa activity detection For the processing and activation methods of sodium citrate anticoagulated plasma samples, please refer to the implementation of coagulation factor IIa activity detection.
  • the test results are shown in Figure 8 (the substances in the box in the legend are pre-mixed). It can be seen from Figure 8 that after pretreatment of plasma with streptokinase, activated coagulation factor Xa caused an enzyme-linked activation reaction.
  • the reaction system contains sodium citrate anticoagulated plasma sample (20 ⁇ l), specific substrate modified proPAO (5 ⁇ l), reaction reagent R1 (90 ⁇ l), reaction reagent R2 (90 ⁇ l).
  • Add the above components to the small wells of the microplate use a microplate reader to shake and mix the reaction system, and detect the changes in absorbance at 555nm and 700nm over time.
  • coagulation factor IIa activity detection Since streptokinase can directly activate plasminogen in plasma to generate plasmin, the activity of fully activated Plasmin in plasma can be directly detected by adding streptokinase.
  • the test results are shown in Figure 9. It can be seen from Figure 9 that Plasminogen in plasma is activated by streptokinase to produce Plasmin, which causes an enzyme-linked activation reaction.
  • the method for detecting the activity of Caspase-3 protein is as follows: add quantitative Caspase-3 and different concentrations of the chromogenic substrate Ac-DEVD-pNA into the wells of the microtiter plate, the reaction system contains Caspase-3 (2 ⁇ l), Ac-DEVD -pNA (2 ⁇ l), 30mM KH 2 PO 4 /Na 2 HPO 4 , pH 8.0 (200 ⁇ l), use a microplate reader to shake and mix the reaction system, and detect the change in absorbance at 405 nm over time.
  • the test results are shown in Figure 10. It can be seen from Figure 10 that under the condition of a certain concentration of Caspase-3, increasing the concentration of the substrate Ac-DEVD-pNA can increase the reaction rate. This reaction can obtain the enzyme activity unit corresponding to a certain quality of Caspase-3.
  • the reaction system includes serum sample (20 ⁇ l), specific substrate modified proPAO (5 ⁇ l), reaction reagent R1 (90 ⁇ l), reaction reagent R2 (90 ⁇ l). Add the above components to the small wells of the microplate, use a microplate reader to shake and mix the reaction system, and detect the changes in absorbance at 555nm and 700nm over time.
  • coagulation factor IIa activity detection For data processing and graphing, please refer to the implementation of coagulation factor IIa activity detection.
  • This reaction can directly detect the activation response of Caspase-3 in the serum to the modified proPAO containing Caspase-3 specific cleavage sequence. The test results are shown in Figure 11. It can be seen from Figure 11 that the enzyme-linked activation reaction rate of Caspase-3 contained in different serum samples to modified proPAO is different.
  • the reaction system contains serum sample (20 ⁇ l), specific substrate modified proPAO (15 ⁇ l), reaction reagent R1 (90 ⁇ l), reaction reagent R2 (90 ⁇ l).
  • Add the above components to the small wells of the microplate use a microplate reader to shake and mix the reaction system, and detect the changes in absorbance at 555nm and 700nm over time.
  • coagulation factor IIa activity detection please refer to the implementation of coagulation factor IIa activity detection.
  • This reaction can directly detect the activation reaction of C5 convertase in serum to the modified proPAO containing the enzyme-specific cleavage sequence.
  • the test results are shown in Figure 12. It can be seen from Figure 12 that the enzyme-linked activation reaction rates of C5 convertase contained in different serum samples to modified proPAO are different.
  • the present invention also selects other zymogens and carries out specific substrate modification to detect the activity of protease in human body.
  • two different sources of polyphenol oxidase proenzymes are selected, namely Prophenoloxidase (signal crayfish Pacifastacus leniusculus) (referred to as proCPO) and Prophenoloxidase 1 (Drosophila melanogaster) (referred to as proDPO).
  • test results showed that the activation effect of the corresponding protease on the modified substrate proCPO only began to appear after about 100 minutes, and the corresponding protease had almost no activation effect on the modified substrate proDPO (as shown in Figure 13). This result indicates that these two recombinant zymogen substrates have lower sensitivity, longer detection time, and are unstable in plasma.
  • the present invention also carried out inserting the specific restriction site sequence only between the ⁇ subunit and the ⁇ subunit.
  • the test process is as follows: to encode the SEQ ID The NO:1 proPAO gene was used as a template, and two pairs of corresponding primers were used to perform overlap extension PCR to obtain a DNA fragment containing a coagulation factor IIa cleavage site (the ⁇ subunit and ⁇ subunit in the pro-phenylalanine oxidase sequence Between), to complete the construction of the specific substrate sequence for coagulation factor IIa activity detection.
  • the zymogen activation reaction cannot be detected when the cleavage site sequence preferred by the protease is not inserted into the phenylalanine oxidase zymogen.
  • the test process is as follows: Using the proPAO gene as a template, three pairs of corresponding primers are used to perform overlap extension PCR to obtain DNA containing a SplB protease cleavage site (WELQ, SEQ ID NO: 68) derived from Staphylococcus aureus Fragments to complete the construction of specific substrate sequences.
  • WELQ SplB protease cleavage site
  • SEQ ID NO:1 Amino acid sequence of phenylalanine oxidase proenzyme from Psuedomonas Sp.P-501
  • the leader peptide sequence is shown in bold double-underlined, the ⁇ subunit is shown in a dashed underline, the ⁇ subunit is shown in a solid underline, and the precursor linker connecting the ⁇ and ⁇ subunits is shown in bold.
  • SEQ ID NO: 2 The amino acid sequence of the phenylalanine oxidase proenzyme from Psuedomonas Sp.P-501 containing two coagulation factor IIa cleavage sites (coagulation factor IIa substrate specific proPAO)
  • the leader peptide sequence is shown with a bold double underline, the ⁇ subunit is shown with a dashed underline, and the ⁇ subunit is shown with a solid underline.
  • the cleavage recognition sequence LRPR of factor IIa is located between the leader peptide sequence and the ⁇ subunit, between the ⁇ subunit and the ⁇ subunit, and is shown in bold italics.
  • SEQ ID NO: 3 NisP pro-enzyme proNisP from Streptococcus lactis containing two coagulation factor Xa cleavage sites (coagulation factor Xa substrate specific proNisP)
  • the leader peptide sequence is double-underlined.
  • the cleavage recognition sequence IEGR of factor Xa is located between the leader peptide sequence and the catalytic subunit, and is shown in bold italics.
  • SEQ ID NO: 4 Pro-phenylalanine oxidase from Psuedomonas Sp.P-501 (NisP substrate specific proPAO) containing two NisP cleavage sites
  • the leader peptide sequence is shown with a bold double underline, the ⁇ subunit is shown with a dashed underline, and the ⁇ subunit is shown with a solid underline.
  • the recognition cleavage sequence ASPRI of NisP is located between the leader peptide sequence and the ⁇ subunit, between the ⁇ subunit and the ⁇ subunit, and is shown in bold italics.
  • SEQ ID NO: 5 Pro-phenylalanine oxidase (Plasmin-specific proPAO) from Psuedomonas Sp.P-501 containing two Plasmin cleavage sites
  • the leader peptide sequence is shown with a bold double underline, the ⁇ subunit is shown with a dashed underline, and the ⁇ subunit is shown with a solid underline.
  • Plasmin's recognition cleavage sequence GYRA is located between the leader peptide sequence and the ⁇ subunit, and between the ⁇ subunit and the ⁇ subunit, shown in bold italics.
  • SEQ ID NO: 6 Pro-phenylalanine oxidase from Psuedomonas Sp.P-501 (Caspase-3 substrate specific proPAO) containing two Caspase-3 cleavage sites
  • the leader peptide sequence is shown with a bold double underline, the ⁇ subunit is shown with a dashed underline, and the ⁇ subunit is shown with a solid underline.
  • the recognition and cleavage sequence DEVD of Caspase-3 is located between the leader peptide sequence and the ⁇ subunit, between the ⁇ subunit and the ⁇ subunit, shown in bold italics.
  • SEQ ID NO: 7 Pro-phenylalanine oxidase from Psuedomonas Sp.P-501 containing two C5 convertase cleavage sites (C5 convertase substrate specific proPAO)
  • the leader peptide sequence is shown with a bold double underline, the ⁇ subunit is shown with a dashed underline, and the ⁇ subunit is shown with a solid underline.
  • the recognition cleavage sequence QLGRLHMK of C5 convertase is located between the leader peptide sequence and the ⁇ subunit, between the ⁇ subunit and the ⁇ subunit, and is shown in bold italics.
  • SEQ ID NO: 8 Specific cleavage sequence of coagulation factor IIa
  • Xaa is any amino acid residue
  • Xab is an amino acid residue preferably selected from Pro, Ala, Gly or Val
  • Xac is an amino acid residue preferably selected from Ser, Ala, Gly
  • SEQ ID NO: 9 Specific cleavage sequence of coagulation factor IIa
  • SEQ ID NO: 10 Specific cleavage sequence of coagulation factor IIa
  • SEQ ID NO: 11 Specific cleavage sequence of coagulation factor IIa
  • SEQ ID NO: 12 Specific cleavage sequence of coagulation factor IIa
  • SEQ ID NO: 13 Specific cleavage sequence of factor VIIa
  • SEQ ID NO: 14 Specific cleavage sequence of coagulation factor IXa
  • SEQ ID NO: 15 Specific cleavage sequence of coagulation factor IXa
  • SEQ ID NO: 16 Specific cleavage sequence of coagulation factor Xa
  • Xaa is any amino acid residue
  • Xad is an amino acid residue preferably selected from Ala or Ile
  • Xae is an amino acid residue preferably selected from Pro, Phe or Gly
  • Xaf is an amino acid residue selected from Arg or Lys Amino acid residue
  • SEQ ID NO: 17 Specific cleavage sequence of coagulation factor Xa
  • SEQ ID NO: 18 Specific cleavage sequence of coagulation factor Xa
  • SEQ ID NO: 19 Specific cleavage sequence of coagulation factor XIa
  • Xag is an amino acid residue preferably from Lys or Asp
  • Xah is an amino acid residue preferably from Phe or Leu
  • SEQ ID NO: 20 Specific cleavage sequence of coagulation factor XIa
  • SEQ ID NO: 21 Specific cleavage sequence of coagulation factor XIIa
  • SEQ ID NO: 22 Specific cleavage sequence of Plasmin
  • Xaa is any amino acid residue
  • Xai is an amino acid residue preferably selected from Arg or Lys
  • Xaj is an amino acid residue preferably selected from Ala, Ser, Gly or Arg
  • SEQ ID NO: 23 Specific cleavage sequence of Plasmin
  • SEQ ID NO: 24 Specific cleavage sequence of Plasmin
  • SEQ ID NO: 25 Caspase-2 specific cleavage sequence
  • SEQ ID NO: 26 Caspase-3 specific cleavage sequence
  • Xak is an amino acid residue preferably from Asp or Glu
  • SEQ ID NO: 27 Caspase-3 specific cleavage sequence
  • SEQ ID NO: 28 Caspase-6 specific cleavage sequence
  • SEQ ID NO: 29 Caspase-7 specific cleavage sequence
  • SEQ ID NO: 30 Caspase-8 specific cleavage sequence
  • Xal is an amino acid residue preferably from Asp or Leu
  • Xam is an amino acid residue preferably from Glu or Ser
  • SEQ ID NO: 31 Caspase-8 specific cleavage sequence
  • SEQ ID NO: 32 Caspase-9 specific cleavage sequence
  • Xaa is any amino acid residue
  • Xan is an amino acid residue preferably selected from Asp or Glu
  • SEQ ID NO: 33 Caspase-10 specific cleavage sequence
  • Xaa is any amino acid residue
  • Xao is an amino acid residue preferably from Glu, Gln or Ser
  • Xap is an amino acid residue preferably from Thr or Val
  • SEQ ID NO: 34 Caspase-14 specific cleavage sequence
  • SEQ ID NO: 35 The specific cleavage sequence of complement pathway C3/C5 convertase
  • SEQ ID NO: 36 Specific cleavage sequence of complement pathway C3/C5 convertase
  • SEQ ID NO: 37 Specific cleavage sequence of MMP-8
  • Xaa is any amino acid residue
  • Xaq is an amino acid residue preferably selected from Pro, Ala or Ser
  • SEQ ID NO: 38 Specific cleavage sequence of MMP-11
  • SEQ ID NO: 39 Specific cleavage sequence of MMP-12
  • Xar is an amino acid residue preferably selected from Pro, Ala or Gly
  • Xas is an amino acid residue preferably selected from Ala or Gly
  • SEQ ID NO: 40 Specific cleavage sequence of MMP-13
  • Xaa is any amino acid residue
  • Xat is an amino acid residue preferably selected from Leu, Ile or Val
  • SEQ ID NO: 41 Specific cleavage sequence of MMP-13
  • SEQ ID NO: 42 Specific cleavage sequence of MMP-20
  • Xaa is any amino acid residue
  • Xau is an amino acid residue preferably from Leu or Met
  • SEQ ID NO: 43 Specific cleavage sequence of MMP-20
  • SEQ ID NO: 44 Specific cleavage sequence of MMP-20
  • SEQ ID NO: 50 Downstream PAO C2-50
  • SEQ ID NO: 56 Upstream PAO B1-56
  • SEQ ID NO: 67 NisP specific identification sequence
  • SEQ ID NO: 68 SplB protease cleavage site

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Abstract

提供了一种经修饰的苯丙氨酸氧化酶酶原，所述修饰是在苯丙氨酸氧化酶酶原序列中的前导序列与α亚基之间以及α亚基与β亚基之间引入蛋白酶的特异性识别位点序列；还提供了所述经修饰的苯丙氨酸氧化酶酶原用于蛋白酶活性检测的用途，其可以对多种蛋白酶的活性进行特异、灵敏和快捷地检测。

Description

经修饰的苯丙氨酸氧化酶酶原及其用途 技术领域
本申请涉及一种经修饰的酶原及其用途，属于蛋白酶活性检测领域。
背景技术
人体内存在多种蛋白酶，然而缺乏一种能够广泛用于检测各种蛋白酶活性的直接、特异、灵敏、快捷、有效的方法。
以凝血系统为例，系统中的因子IIa也被称为凝血酶，属于丝氨酸蛋白酶，为血液促凝系统中血栓与止血的关键环节。循环血液中的凝血酶以无活性的凝血酶原形式存在，正常人体内仅含有极微量的有活性的凝血酶。目前，凝血酶的检测方法大致分为间接检测法，包含测定凝血酶原片段1+2(F1+2)、纤维蛋白肽A、可溶性纤维蛋白单体复合物、凝血酶-抗凝血酶复合物、凝血活酶生成等；直接检测法，包含发色底物法与荧光法。然而，由于激活状态与非激活状态的凝血因子间在氨基酸序列上仅有十几至几十个的数目差异，上述办法均难以对其进行区分。此外，目前凝血酶的间接检测法应用较为广泛，但其敏感性和特异性相对较差。目前还未见有直接检测血液中生理状态和激活状态的凝血因子IIa活性的相关报道。
体内的纤溶系统和凝血系统紧密联系，纤溶系统中的纤溶酶原(Plasminogen)被链激酶或尿激酶激活后成为纤溶酶(Plasmin)，后者可专一降解纤维蛋白凝胶，使其被分解成可溶性产物，通过负反馈效应降低体内纤维蛋白原的水平，从而避免纤维蛋白的过多凝聚。目前还未见有直接检测血液中生理状态和激活状态的纤溶酶活性的相关报道。
凝血象的检测结果反应的是凝血系统的一种整体表现，很难定位到某一个凝血因子的情况。而关于现有的检测凝血因子的ELISA方法，只是检测特定蛋白是否存在及其含量，而并没有反应酶的活性。此外，该方法中的抗体效价及特异性问题限制了检测结果的标准化。
目前仍需要一种能够广泛用于人体内各种蛋白酶活性检测的直接、灵敏、快捷、特异、有效的方法。
发明概述
一方面，本发明提供一种经修饰的苯丙氨酸氧化酶酶原(the proenzyme of L-phenylalanine oxidase，简写为proPAO)，其特征在于：所述修饰是在苯丙氨酸氧化酶酶原序列中的前导序列与α亚基间以及α亚基与β亚基间引入有蛋白酶的特异性识别位点序列。
在一些实施方案中，苯丙氨酸氧化酶酶原来源于细菌，例如Pseudomonas Sp.P-501，东湖假单胞菌(Pseudomonas donghuensis)，茄科雷尔氏菌(Ralstonia solanacearum)UW551或洋葱伯克氏菌(Burkholderia cepacia)ATCC 25416。
在一些实施方案中，蛋白酶选自凝血级联蛋白酶，例如凝血因子IIa、Va、VIIa、VIIIa、IXa、Xa、XIa或XIIa；纤溶酶；含半胱氨酸的天冬氨酸蛋白酶，例如Caspase-1、Caspase-2、Caspase-3、Caspase-4、Caspase-5、Caspase-6、Caspase-7、Caspase-8、Caspase-9、Caspase-10或Caspase-11；补体途径蛋白酶，例如因子C1、C2、C3、C4、C5、C6、C7、C8、C9或C3/C5转换酶；MMP家族蛋白酶；NisP酶。
技术人员知晓如何确定蛋白酶的特异性识别位点序列，且熟知本领域已知的蛋白酶的特异性识别位点序列。
在一些实施方案中，所述蛋白酶的特异识别位点序列如SEQ ID NO:8-44、67任一所示，例如SEQ ID NO:9-12任一所示。
例如，凝血因子IIa的切割序列包括但不限于Xaa Xaa Xab Arg Xac(SEQ ID NO:8)，切割位点在Arg与Xac之间，其中“Xaa”为任意氨基酸残基，“Xab”为优选自Pro、Ala、Gly或Val的氨基酸残基，“Xac”为优选自Ser、Ala、Gly的氨基酸残基，优选Leu Val Pro Arg Gly(SEQ ID NO:10)；凝血因子IIa的切割序列也可以为Leu Arg Pro Arg(SEQ ID NO:9)(切割位点在Arg之后)、Phe Pro Arg(SEQ ID NO:11)(切割位点在Arg之后)和Gly Arg Gly(SEQ ID NO:12)(切割位点在Arg与Gly之间)等。凝血因子VIIa的切割序列包括但不限于Leu Ile Gln Arg(SEQ ID NO:13)。凝血因子IXa的切割序列包括但不限于Xaa Xaa Gly Arg(SEQ ID NO:14)，“Xaa”为任意氨基酸残基，优选Pro Gln Gly Arg(SEQ ID NO:15)。凝血因子Xa的切割序列包括但不限于Xad Xaa Xae Xaf(SEQ ID NO:16)，其中“Xaa”为任意氨基酸残基，“Xad”为优选自Ala或Ile的氨基酸残基，“Xae”为优选自Pro、Phe或Gly的氨基酸残基，“Xaf”为选自Arg或 Lys的氨基酸残基，优选Ile Glu Gly Arg(SEQ ID NO:17)和Ile Asp Gly Arg(SEQ ID NO:18)等。凝血因子XIa的切割序列包括但不限于Xag Xah Thr Arg(SEQ ID NO:19)，其中“Xag”为优选自Lys或Asp的氨基酸残基，“Xah”为优选自Phe或Leu的氨基酸残基，优选Lys Leu Thr Arg(SEQ ID NO:20)。凝血因子XIIa的切割序列包括但不限于Thr Ser Thr Arg(SEQ ID NO:21)。纤溶酶Plasmin的切割序列包括但不限于Xaa Xaa Xai Xaj(SEQ ID NO:22)，切割位点在Xai与Xaj之间，其中“Xaa”为任意氨基酸残基，“Xai”为优选自Arg或Lys的氨基酸残基，“Xaj”为优选自Ala、Ser、Gly或Arg的氨基酸残基，优选Gly Tyr Arg Ala(SEQ ID NO:23)和Pro Ala Lys Ala(SEQ ID NO:24)。Caspase-2的切割序列包括但不限于Asp Glu Xaa Asp(SEQ ID NO:25)，其中“Xaa”为任意氨基酸残基。Caspase-3的切割序列包括但不限于Xak Glu Val Asp(SEQ ID NO:26)，其中“Xak”为优选自Asp或Glu的氨基酸残基，优选Asp Glu Val Asp(SEQ ID NO:27)。Caspase-6的切割序列包括但不限于Xaa Glu Xaa Asp(SEQ ID NO:28)，其中“Xaa”为任意氨基酸残基。Caspase-7的切割序列包括但不限于Asp Xaa Xaa Asp(SEQ ID NO:29)，其中“Xaa”为任意氨基酸残基。Caspase-8的切割序列包括但不限于Xal Xam Thr Asp(SEQ ID NO:30)，其中“Xal”为优选自Asp或Leu的氨基酸残基，“Xam”为优选自Glu或Ser的氨基酸残基，优选Asp Glu Thr Asp(SEQ ID NO:31)。Caspase-9的切割序列包括但不限于Xaa Xan Xaa Asp(SEQ ID NO:32)，其中，“Xaa”为任意氨基酸残基，“Xan”为优选自Asp或Glu的氨基酸残基。Caspase-10的切割序列包括但不限于Xaa Xao Xap Asp(SEQ ID NO:33)，其中，“Xaa”为任意氨基酸残基，“Xao”为优选自Glu、Gln或Ser的氨基酸残基，“Xap”为优选自Thr或Val的氨基酸残基。Caspase-14的切割序列包括但不限于Leu Glu Xaa Asp(SEQ ID NO:34)，其中“Xaa”为任意氨基酸残基。补体途径C3/C5转化酶的切割序列包括但不限于Gln Leu Gly Arg Leu His Met Lys(SEQ ID NO:35)(切割位点在Arg与Leu之间)和Gly Leu Ala Arg Ser Asn Leu Asp(SEQ ID NO:36)(切割位点在Arg与Ser之间)。MMP-8的切割序列包括但不限于Gly Xaq Xaa Gly(SEQ ID NO:37)，其中，“Xaa”为任意氨基酸残基，“Xaq”为优选自Pro、Ala或Ser的氨基酸残基。MMP-11的切割序列包括但不限于Xaa Ala Ala Ala(SEQ ID NO:38)，其中“Xaa”为任意氨基酸残基。MMP-12的切割序列包括但不限于 Gly Xar Xas Xas(SEQ ID NO:39),其中“Xar”为优选自Pro、Ala或Gly的氨基酸残基,“Xas”为优选自Ala或Gly的氨基酸残基。MMP-13的切割序列包括但不限于Gly Pro Xaa Gly Xat(SEQ ID NO:40)(切割位点在Gly与Xat之间)，其中“Xaa”为任意氨基酸残基，“Xat”为优选自Leu、Ile或Val的氨基酸残基，优选Gly Pro Ala Gly Leu(SEQ ID NO:41)。MMP-20的切割序列包括但不限于Pro Xaa Leu Pro Xau(SEQ ID NO:42)(切割位点在Pro与Xau之间)，其中“Xaa”为任意氨基酸残基，“Xau”为优选自Leu或Met的氨基酸残基，优选Pro Ala Leu Pro Leu(SEQ ID NO:43)或Pro Ala Leu Pro Met(SEQ ID NO:44)。
在一些实施方案中，所述苯丙氨酸氧化酶酶原序列如SEQ ID NO:1所示。
在一些实施方案中，所述经修饰的苯丙氨酸氧化酶酶原序列如SEQ ID NO:2、4-7任一项所述。
在第二方面，本发明提供了第一方面所述经修饰的苯丙氨酸氧化酶酶原用于检测样品中蛋白酶活性的用途。
在该方面，本发明还提供了检测样品中蛋白酶活性的方法，所述方法包括使第一方面所述经修饰的苯丙氨酸氧化酶酶原与所述样品接触。
在本发明中，所述样品可以是体液、全血、血浆、血清或组织。
在凝血因子IIa活性检测中，以来源于Pseudomonas Sp.P-501的苯丙氨酸氧化酶酶原为反应底物，通过基因工程技术在其中引入凝血因子IIa的特异识别序列，利用苯丙氨酸氧化酶(PAO)与底物的氧化还原与显色反应，构建凝血因子IIa浓度与反应产物吸光度变化间的时间曲线，建立凝血因子IIa活性的直接检测体系。同时，使用临床样本对检测体系进行可行性验证，确认检测方法适用于枸橼酸钠抗凝血浆样本的检测。本发明构建了完整的凝血因子IIa检测方法，通过凝血因子IIa、氧化还原酶两级级联反应，拓宽了检测范围、提高了检测灵敏度。
在低含量待测蛋白酶的实施方案中，如对于凝血因子Xa，运用该检测体系需要增加一步酶原激活级联反应以放大Xa因子的浓度信号，例如中间蛋白酶原满足基本的放大要求，即不存在于人体内，识别序列特异性强，反应pH值呈中性，可以于30分钟内检测出血浆样本的吸光度变化。
在一些实施方案中，中间蛋白酶原是经修饰的NisP蛋白酶酶原(proNisP)，其在前导肽和催化亚基之间包含酶特异性识别位点序列。例如，经修饰的 proNisP来自乳酸链球菌。
在凝血因子Xa和IIa的活性检测过程中，运用链激酶对血浆进行预处理,可防止全激活状态时血浆样本的凝固。
在纤溶酶酶活性的检测中，纤溶酶原可以被链激酶直接激活生成纤溶酶，后者引起酶联激活反应，从而建立纤溶酶活性的直接检测体系。
此外，使用枸橼酸钠抗凝的血浆样本对凝血因子和纤溶酶进行了可行性验证，比较了血浆在生理状态和全激活状态下的各蛋白酶活性。
在Caspase-3酶活性的检测中，以苯丙氨酸氧化酶酶原为反应底物，通过基因工程技术构建入Caspase-3的特异识别序列，利用PAO与底物的氧化还原与显色反应，建立Caspase-3活性的直接检测体系。同时，使用临床样本对检测体系进行可行性验证，确认检测方法适用于血清样本的检测。
在C5转换酶酶活性的检测中，以苯丙氨酸氧化酶酶原为反应底物，通过基因工程技术构建入C5转换酶的特异识别序列，利用PAO与底物的氧化还原与显色反应，建立补体系统中C5转换酶活性的直接检测体系。同时，使用临床样本对检测体系进行可行性验证，确认检测方法适用于血清样本的检测。
在一些实施方案中，采用的血浆标本为枸橼酸钠抗凝血浆样本或肝素抗凝血浆样本。
在一些实施方案中，还包括采用链激酶或尿激酶预处理样品。
在一些实施方案中，包括激活的苯丙氨酸氧化酶与底物的氧化还原与显色反应。
在第三方面，本发明提供了一种分离的核酸，其编码第一方面所述的经修饰的苯丙氨酸氧化酶酶原。
在第四方面，本发明提供了一种表达载体，其包含第三方面所述的分离的核酸。
在第五方面，本发明提供了用第四方面所述的表达载体转化或转染的宿主细胞。
第六方面，本发明提供了一种检测样品中蛋白酶活性的试剂盒，其包括第一方面所述的经修饰的苯丙氨酸氧化酶酶原。
在一些实施方案中，所述试剂盒还包含经修饰的proNisP，其在前导肽 和催化亚基之间包含酶特异性识别位点序列，例如凝血因子Xa的特异识别序列。
来自Psuedomonas Sp.P-501的没有催化活性的L-苯丙氨酸氧化酶酶原(proPAO)基本由14个氨基酸残基的前导序列、92个氨基酸残基的α亚基、一个二肽(Ile-Lys)和605个残基的β亚基组成。根据已有文献中体外蛋白酶水解proPAO的实验结果，proPAO可以被蛋白酶Pronase和Trypsin共同水解成缺失第一个赖氨酸残基的α亚基和β亚基片段，该水解产物的活性与含有α亚基和β亚基片段的活化形式的PAO活性相当(Suzuki,H.et al.Sequencing and expression of the L-phenylalanine oxidase gene from Pseudomonas sp.P-501.Proteolytic activation of the proenzyme.Journal of biochemistry 136,617-627,doi:10.1093/jb/mvh169(2004)；Ida,K.et al.Structural basis of proteolytic activation of L-phenylalanine oxidase from Pseudomonas sp.P-501.The Journal of biological chemistry 283,16584-16590,doi:10.1074/jbc.M800366200(2008)；Ida,K.,Suguro,M.& Suzuki,H.High resolution X-ray crystal structures of L-phenylalanine oxidase(deaminating and decarboxylating)from Pseudomonas sp.P-501.Structures of the enzyme-ligand complex and catalytic mechanism.Journal of biochemistry 150,659-669,doi:10.1093/jb/mvr103(2011))。因此，本文中所称的α亚基包括了N端缺少一个赖氨酸的形式，此时所称前导序列则可以对应地在C端多一个赖氨酸。
定义
苯丙氨酸氧化酶：指苯丙氨酸2-单加氧酶(EC 1.13.12.9)，其主要催化L-苯丙氨酸生成2-苯基乙酰胺或3-苯丙酮酸，其底物还包括β-2-噻吩丙氨酸，L-酪氨酸、L-甲硫氨酸等。
酶原：具有酶的特定活性的酶的前体形式，由此一般称之为无活性形式的酶。本文所称的酶原可以指在检测方法中经酶切活化成有活性的酶的前体形式。酶原可通过导致活化级联的其他酶活化成对应活性酶。
修饰：在多肽(例如酶)中的一个或多个氨基酸残基的突变(置换)、插入(添加)或缺失，以获得修饰的多肽。本文中，术语“重组”、“修饰”、“改造”可互换使用。
识别位点序列：是指检测中被酶所特异性识别并切割的氨基酸序列。本 文中，术语“识别位点序列”、“识别序列”或“切割序列”可互换使用。
蛋白酶酶原：可活化成活性蛋白酶的蛋白酶前体形式。本文所称的蛋白酶包括丝氨酸蛋白酶，苏氨酸蛋白酶，半胱氨酸蛋白酶，天冬氨酸蛋白酶，金属蛋白酶或谷氨酸蛋白酶等任意可切割蛋白序列的酶。
氨基酸残基：包括但不限于由以下氨基酸组成的集合的氨基酸残基：丙氨酸(三字母代码：Ala，单字母代码：A)，精氨酸(Arg，R)，天冬酰胺(Asn，N)，天冬氨酸(Asp，D)，半胱氨酸(Cys，C)，谷氨酰胺(Gln，Q)，谷氨酸(Glu，E)，甘氨酸(Gly，G)，组氨酸(His，H)，异亮氨酸(Ile，I)，亮氨酸(Leu，L)，赖氨酸(Lys，K)，甲硫氨酸(Met，M)，苯丙氨酸(Phe，F)，脯氨酸(Pro，P)，丝氨酸(Ser，S)，苏氨酸(Thr，T)，色氨酸(Trp，W)，酪氨酸(Tyr，Y)，和缬氨酸(Val，V)。
编码序列：编码序列的边界一般由开放阅读框架确定，该开放阅读框架从起始密码子(如ATG、GTG或TTG)开始并且以终止密码子(如TAA、TAG或TGA)结束。编码序列可以是基因组DNA、cDNA、合成DNA或其组合。
转化：指将DNA导入受体宿主细胞，其改变基因型并随后导致受体细胞中的改变。
宿主细胞：指经过使用重组DNA技术构建且编码至少一种异源基因的载体转化的细胞。
酶活性检测方法：
A.凝血因子IIa
1.凝血因子IIa特异底物(即包含凝血因子IIa特异识别位点序列的经修饰苯丙氨酸氧化酶酶原)的制备方法
凝血因子IIa特异底物的制备方法，包括菌株、质粒、酶与培养基，PCR扩增及重组质粒的构建，基因的诱导表达与蛋白质的纯化。具体步骤如下：
(1)菌株、质粒、酶与培养基：
大肠杆菌(Escherichia coli)DH5α、BL21(DE3)、克隆与表达质粒载体pRSFDuet-1购自Novagen公司；DNA高保真聚合酶混合液购自Tsingke公司；限制性内切酶、DNA连接酶购自NEB公司；大肠杆菌LB培养基：每升含Tryptone 10g，Yeast extract 5g，NaCl 10g，卡那霉素浓度为50mg/L。
(2)重叠延伸PCR扩增及重组质粒的构建：
全基因合成编码proPAO(即SEQ ID NO:1)的基因序列，设计三对引物，通过重叠延伸PCR在酶原proPAO的激活位点处(即前导序列与α亚基间，和α亚基与β亚基间)加入两个凝血因子IIa的特异识别序列(氨基酸序列LRPR，SEQ ID NO:9)。经限制性内切酶双酶切连接至经同样内切酶酶切的表达载体pRSFDuet-1上，连接产物转化大肠杆菌E.coli DH5α，筛选重组质粒，并进行双酶切及测序验证。
上述重叠延伸PCR的引物如下：
上游PAO A1-45:
5′-catg ccatggtgggcgttaccgtcattccccggctgc-3′(SEQ ID NO:45)
下游PAO A2-46:
5′-gcccgcgtaccttgatcgcacgtggacgcagtcccgggcggtcatgaaga-3′(SEQ ID NO:46)
上游PAO B1-47:
5′-tcttcatgaccgcccgggactgcgtccacgtgcgatcaaggtacgcgggc-3′(SEQ ID NO:47)
下游PAO B2-48:
5′-ggaattc catatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′(SEQ ID NO:48)
上游PAO C1-49:
5′-cggcctgaaggacgagaagctgcgtccacgtaagattgccaccaccgttg-3′(SEQ ID NO:49)
下游PAO C2-50:
5′-caacggtggtggcaatcttacgtggacgcagcttctcgtccttcaggccg-3′(SEQ ID NO:50)
其中，划线部分为修饰后的proPAO上游引物NcoI酶切位点及下游引物NdeI酶切位点。限制性内切酶选用NcoI和NdeI两种。
(3)基因的诱导表达与蛋白质的纯化：
将重组质粒转化E.coli BL21(DE3)。加入IPTG于培养物中进行诱导表达。诱导表达的蛋白经过超声破壁，离心，取上清液进行镍柱亲和层析(QIAGEN公司产品)和脱盐层析柱(Amersham公司产品)得到纯化的凝血因子IIa底物特异性的proPAO。
(4)凝血因子IIa酶解底物的验证
取商品化凝血因子IIa纯品(Sigma公司产品)，加入至上述纯化的凝血因子IIa底物特异性proPAO蛋白溶液，37℃孵育，SDS-PAGE验证水解效率。
2.凝血因子IIa活性检测方法的建立
(1)凝血因子IIa对其底物的酶解
凝血因子IIa底物特异性proPAO经凝血因子IIa催化水解后，α与β亚基被释放出，PAO的结构不受影响，具有催化活性。
(2)PAO与底物的氧化还原反应与显色反应
反应过程可分为二步：
Figure PCTCN2020083317-appb-000001
Figure PCTCN2020083317-appb-000002
首先，PAO催化底物L-苯丙氨酸(L-Phe)的氧化脱氨基反应，生成β-苯丙酮酸(β-phenylpyruvate)、氨(NH 3)和过氧化氢(H 2O 2)。接着根据Trinder反应，在辣根过氧化物酶(HRP)的催化作用下，过氧化氢(H 2O 2)与酚衍生物(TOOS)、4-氨基安替比林(4-AAT)反应生成醌类显色化合物(Quinones)，该化合物为红紫色，最大吸收峰位于550-560nm处，颜色强度与过氧化氢的浓度成正比。为消除检测过程中的随机误差，本体系的信号检测选取双波长分光光度法。以最大吸收峰处波长550-560nm为测定波长，以可见光范围内吸收最小处的波长700nm为组合波长。以Abs 555nm-Abs 700nm处理吸光度检测数据，作为检测的分析信号。
(3)酶活性检测条件的优化
酶反应速率随底物proPAO浓度的升高而加快。然而，高浓度的proPAO将造成底物的空间距离过近，导致较高的proPAO背景反应。故选择反应速率相对较高的proPAO浓度为检测反应的终浓度。同时对其他底物(L-Phe、HRP、TOOS和4-AAT)浓度及反应pH值分别进行优化，使得反应体系有最大吸光度变化速率。
3.临床标本的检测
采用临床枸橼酸钠抗凝的血浆标本，在反应体系中补充足量的钙离子(CaCl 2)和组织因子(Tissue Factor，TF)以充分激活待测血浆中的凝血因子IIa。为去除标本中的纤维蛋白原以阻止标本凝固，采用链激酶(Streptokinase，SK)预先处理标本以降解其中的纤维蛋白，再对处理过的标本进行检测，可以防止反应体系凝固。
B.凝血因子Xa
在人体凝血系统中，凝血因子Xa位于凝血因子IIa的上游，本发明中通过选择中间酶原增加一步酶原激活反应以放大Xa因子的浓度信号。具体选择了来源于乳酸链球菌(Streptococcus lactis)的NisP酶酶原(Nisin leader peptide-processing serine protease proenzyme，proNisP)。全基因合成重组的proNisp基因序列，在该酶原的前导肽序列与催化亚基之间加入Xa因子的特异识别序列IEGR(SEQ ID NO:17)。接着设计三对引物，通过重叠延伸PCR在proPAO的激活位点处加入两个NisP的特异识别序列ASPRI(SEQ ID NO:67)。
上述重叠延伸PCR的引物如下：
上游PAO A1-45:
5′-catg ccatggtgggcgttaccgtcattccccggctgc-3′(SEQ ID NO:45)
下游PAO A2-51:
5′-gcccgcgtaccttgatcgcaatacgaggagatgctcccgggcggtcatga-3′(SEQ ID NO:51)
上游PAO B1-52:
5′-tcatgaccgcccgggagcatctcctcgtattgcgatcaaggtacgcgggc-3′(SEQ ID NO:52)
下游PAO B2-48:
5′-ggaattc catatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′(SEQ ID NO:48)
上游PAO C1-53:
5′-cctgaaggacgagaaggcatctcctcgtattaagattgccaccaccgttg-3′(SEQ ID NO:53)
下游PAO C2-54:
5′-caacggtggtggcaatcttaatacgaggagatgccttctcgtccttcagg-3′(SEQ ID NO:54)
其中，划线部分为改造后的proPAO上游引物NcoI酶切位点及下游引物NdeI酶切位点。限制性内切酶选用NcoI和NdeI两种。
将构建好的重组酶原proNisp和proPAO分别连接至表达载体pRSFDuet-1并转化入E.coli BL21(DE3)，分别进行基因的诱导表达与蛋白质的纯化。proNisp的诱导表达与蛋白质的纯化如下：加入IPTG于培养物中进行诱导表达。诱导表达的蛋白经过超声破壁，离心，取上清液进行镍柱亲和层析(QIAGEN公司产品)和脱盐层析柱(Amersham公司产品)得到蛋白产物。
取商品化凝血因子Xa纯品(NEB公司产品)和纯化的重组proNisP酶原加入至上述的重组proPAO，37℃孵育，SDS-PAGE验证水解效率。
临床标本的凝血因子Xa活性检测：采用临床枸橼酸钠抗凝的血浆标本，在反应体系中补充足量的CaCl 2和TF以充分激活待测血浆中的凝血因子Xa。为去除标本中的纤维蛋白原以阻止标本凝固，采用链激酶预先处理标本以降解其中的纤维蛋白，再对处理过的标本进行检测。
C.纤溶酶
以proPAO(Pseudomonas Sp.P-501)基因为模板，设计三对引物，通过重叠延伸PCR在酶原proPAO的激活位点处加入两个Plasmin的特异识别序列GYRA(SEQ ID NO:23)。经限制性内切酶双酶切连接至经同样内切酶酶切的表达载体pRSFDuet-1上，连接产物转化大肠杆菌E.coli DH5α，筛选重组质粒，并进行双酶切及测序验证。
上述重叠延伸PCR的引物如下：
上游PAO A1-45:
5′-catg ccatggtgggcgttaccgtcattccccggctgc-3′(SEQ ID NO:45)
下游PAO A2-55:
5′-cagcccgcgtaccttgatcgcacgataacctcccgggcggtcatgaagaa-3′(SEQ ID NO:55)
上游PAO B1-56:
5′-ttcttcatgaccgcccgggaggttatcgtgcgatcaaggtacgcgggctg-3′(SEQ ID NO:56)
下游PAO B2-48:
5′-ggaattc catatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′(SEQ ID NO:48)
上游PAO C1-57:
5′-cggcctgaaggacgagaagggttatcgtgcaaagattgccaccaccgttg-3′(SEQ ID NO:57)
下游PAO C2-58:
5′-caacggtggtggcaatctttgcacgataacccttctcgtccttcaggccg-3′(SEQ ID NO:58)
其中，划线部分为改造后的proPAO上游引物NcoI酶切位点及下游引物NdeI酶切位点。限制性内切酶选用NcoI和NdeI两种。
后续的实施内容可以参照凝血因子IIa活性检测方法，需注意，与凝血因子IIa和Xa不同的是，在纤溶酶活性检测方法的实施方案中，链激酶可以 直接激活血浆中的纤溶酶原使之生成纤溶酶，后者引起酶联激活反应，从而建立纤溶酶活性的直接检测体系。
D.Caspase-3
以proPAO(Pseudomonas Sp.P-501)基因为模板，设计三对引物，通过重叠延伸PCR在酶原proPAO的激活位点处加入两个Caspase-3的特异识别序列DEVD(SEQ ID NO:27)。经限制性内切酶双酶切连接至经同样内切酶酶切的表达载体pRSFDuet-1上，连接产物转化大肠杆菌E.coli DH5α，筛选重组质粒，并进行双酶切及测序验证。
上述重叠延伸PCR的引物如下：
上游PAO A1-45:
5′-catg ccatggtgggcgttaccgtcattccccggctgc-3′(SEQ ID NO:45)
下游PAO A2-59:
5′-gcccgcgtaccttgatcgcatcaacttcgtctcccgggcggtcatgaaga-3′(SEQ ID NO:59)
上游PAO B1-60:
5′-tcttcatgaccgcccgggagacgaagttgatgcgatcaaggtacgcgggc-3′(SEQ ID NO:60)
下游PAO B2-48:
5′-ggaattc catatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′(SEQ ID NO:48)
上游PAO C1-61:
5′-cggcctgaaggacgagaaggacgaagttgataagattgccaccaccgttg-3′(SEQ ID NO:61)
下游PAO C2-62:
5′-caacggtggtggcaatcttatcaacttcgtccttctcgtccttcaggccg-3′(SEQ ID NO:62)
其中，划线部分为改造后的proPAO上游引物NcoI酶切位点及下游引物NdeI酶切位点。限制性内切酶选用NcoI和NdeI两种。
Caspase-3的酶活力单位：Caspase-3可水解乙酰基-Asp-Glu-Val-Asp-对硝基苯胺(Ac-DEVD-pNA)，释放对硝基苯胺(pNA)部分，可在405nm处测定。通过检测重组Caspase-3对显色底物Ac-DEVD-pNA的切割活性可以定义Caspase-3的酶活力单位。
后续底物proPAO激活反应的实施内容可以参照凝血因子IIa活性检测方法，需注意，与凝血系统中的检测方法不同的是，在所有的Caspase活性检 测方法的实施方案中，采用的临床标本为血清成分。此外，反应中不需要使用激活剂，可以直接通过检测血清中Caspase-3对含有特异切割序列的重组酶原proPAO的激活反应，建立Caspase-3活性的直接检测体系。
E.C5转化酶
以proPAO(Pseudomonas Sp.P-501)基因为模板，设计三对引物，通过重叠延伸PCR在酶原proPAO的激活位点处加入两个C5转化酶的特异识别序列QLGRLHMK(SEQ ID NO:35)。经限制性内切酶双酶切连接至经同样内切酶酶切的表达载体pRSFDuet-1上，连接产物转化大肠杆菌E.coli DH5α，筛选重组质粒，并进行双酶切及测序验证。
上述重叠延伸PCR的引物如下：
上游PAO A1-45:
5′-catg ccatggtgggcgttaccgtcattccccggctgc-3′(SEQ ID NO:45)
下游PAO A2-63:
5′-cgcgtaccttgatcgctttcatgtgcagacgacccagttgtcccgggcggtcatga-3′(SEQ ID NO:63)
上游PAO B1-64:
5′-tcatgaccgcccgggacaactgggtcgtctgcacatgaaagcgatcaaggtacgcg-3′(SEQ ID NO:64)
下游PAO B2-48:
5′-ggaattc catatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′(SEQ ID NO:48)
上游PAO C1-65:
5′-cctgaaggacgagaagcaactgggtcgtctgcacatgaagattgccaccaccgttg-3′(SEQ ID NO:65)
下游PAO C2-66:
5′-caacggtggtggcaatcttcatgtgcagacgacccagttgcttctcgtccttcagg-3′(SEQ ID NO:66)
其中，划线部分为改造后的proPAO上游引物NcoI酶切位点及下游引物NdeI酶切位点。限制性内切酶选用NcoI和NdeI两种。
后续的实施内容可以参照Caspase-3活性检测方法，可以直接通过检测 血清中C5转化酶对含有特异切割序列的重组酶原proPAO的激活反应，建立补体系统中C5转化酶活性的直接检测体系。
附图说明
图1为凝血因子IIa特异底物proPAO(含LRPR识别序列)纯化产物的SDS-PAGE检测结果，其中：M是蛋白marker。
图2为凝血因子IIa对特异底物proPAO(含LRPR识别序列)酶解的SDS-PAGE检测结果，其中：M是蛋白marker。
图3为凝血因子IIa纯品对因子IIa特异底物proPAO(含LRPR识别序列)的酶解活性定量检测结果。
图4为凝血因子IIa活力单位与对应反应速率方程参数间的线性关系。
图5为链激酶预处理后血浆中的凝血因子IIa对底物proPAO(含LRPR识别序列)的酶解活性检测结果。
图6为含有两个凝血因子Xa切割位点的proNisP(含IEGR识别序列)和NisP特异性底物proPAO(含ASPRI识别序列)纯化产物的SDS-PAGE检测结果，其中：20、250分别是镍柱亲和层析中咪唑的洗脱浓度(单位mM)，Des是脱盐层析，M是蛋白marker。
图7为凝血因子Xa酶解因子Xa特异底物proNisP(含IEGR识别序列)后的产物NisP，以及产物NisP对NisP特异底物proPAO(含ASPRI识别序列)酶解的SDS-PAGE检测结果，其中：M是蛋白marker。
图8为链激酶预处理后血浆中的凝血因子Xa通过酶解因子Xa特异底物proNisP(含IEGR识别序列)对NisP特异底物proPAO(含ASPRI识别序列)的酶联水解活性检测结果。
图9为链激酶激活纤溶酶原后血浆中的纤溶酶对特异底物proPAO(含GYRA识别序列)的酶解活性检测结果。
图10为重组Caspase-3对合成底物Ac-DEVD-pNA的切割活性检测结果。
图11为不同受检者血清中Caspase-3对特异底物proPAO(含DEVD识别序列)的酶解活性检测结果。其中：Sample 1为小细胞肺癌和肝转移患者，Sample 2为贲门病变患者，Sample 3为疑似淋巴瘤患者，Sample 4为体检者。
图12为不同受检者血清中C5Convertase对特异底物proPAO(含 QLGRLHMK识别序列)的酶解活性检测结果。其中：Sample 1与Sample 2为风湿免疫疾病患者，Sample 3与Sample 4为体检者。
图13中A和B分别为特异底物多酚氧化酶原proCPO(signal crayfish Pacifastacus leniusculus)和proDPO(Drosophila melanogaster)被凝血因子激活的检测结果。
图14为链激酶预处理后血浆中的凝血因子IIa对底物proPAO(含一个LRPR识别序列)的酶解活性检测结果。
图15为链激酶预处理后血浆中的凝血因子IIa对来源于金黄色葡萄球菌(Staphylococcus aureus)的SplB蛋白酶特异底物proPAO(含WELQ，SEQ ID NO:68识别序列)的酶解活性检测结果。
图16为经特定蛋白酶识别序列修饰的proPAO的酶原激活反应测定相应蛋白酶活性的流程图。
具体实施方式
蛋白酶活性检测的具体实施例包含凝血级联蛋白酶(包含多种凝血因子)，纤溶酶，含半胱氨酸的天冬氨酸蛋白酶(包含Caspase家族成员)，补体途径蛋白酶(包含各个补体成分)和基质金属蛋白酶(包含MMP家族成员)。
实施例1.凝血因子IIa活性检测
1.凝血因子IIa底物特异性的proPAO序列的构建
(1)扩增获得含有一个凝血因子IIa切割位点的proPAO DNA片段
分别设计引物PAO A1-45上游和PAO A2-46下游，PAO B1-47上游和PAO B2-48下游，引物PAO A1-45上游和PAO B2-48下游中分别加入了NcoI和NdeI酶切位点。全基因合成Pseudomonas Sp.P-501中的proPAO序列(编码序列为SEQ ID NO:1)，以此DNA序列为模板，用引物PAO A1-45上游和PAO A2-46下游，PAO B1-47上游和PAO B2-48下游分别扩增含有一个凝血因子IIa切割位点(位于α亚基与β亚基之间)的两部分DNA片段A和B。PCR反应体系：1.1×PCR mix buffer 20μl，上游、下游引物(10μM)各1μl，模板0.2μl。反应条件：94℃热变性5min；94℃变性45s，60℃退火45s，72℃延伸30s(使用引物PAO A1和PAO A2时)或2min(使用引物PAO B1和PAO B2时)，共30个循环；72℃延伸10min。PCR产物在0.5×TAE配制的琼 脂糖凝胶中电泳，DNA回收试剂盒(Tiangen公司)回收目的片段，方法参考其说明书，回收产物-20℃保存备用。
将使用引物PAO A1-45和PAO A2-46扩增得到的A片段与使用引物PAO B1-47和PAO B2-48得到的B片段进行重叠延伸PCR。引物使用PAO A1-45上游和PAO B2-48下游。PCR反应体系：1.1×PCR mix buffer 20μl，上游、下游引物(10μM)各1μl，模板A片段与B片段各1μl。反应条件：94℃热变性5min；94℃变性45s，60℃退火45s，72℃延伸2min 10s，共30个循环；72℃延伸10min。琼脂糖凝胶中电泳回收PCR产物同前。得到纯的含有一个凝血因子IIa切割位点(位于α亚基与β亚基之间)的DNA片段。
(2)克隆含有一个凝血因子IIa切割位点的proPAO DNA片段
对回收得到的DNA片段进行限制性内切酶消化，反应体系：回收的PCR产物43μl，10×限制性内切酶缓冲液5μl，NcoI(Thermal Scientific公司)1μl，NdeI(Thermal Scientific公司)1μl。混匀，37℃过夜酶解。通过琼脂糖电泳回收经过酶切的PCR扩增基因，与经过同样的两个限制性内切酶酶切回收得到的质粒载体pRSFDuet-1相连。连接反应体系：经过酶切处理的PCR扩增基因12μl，经过酶切处理的质粒载体2μl，10×T4连接酶缓冲液2μl，T4连接酶(Thermal Scientific公司)0.2μl，ddH 2O 3.8μl。混匀，20℃反应2h。
连接产物的转化：取100μl感受态细胞E.coli DH5α于1.5ml离心管中，加入10μl连接产物。冰上放置30min，42℃热激90s，冰上放置2min。加入500μl LB培养基，37℃振荡培养45min，使细菌复苏。
转化菌的培养：12000rpm离心1min，弃上清，用100μl LB培养基重悬菌体，涂布于卡那霉素抗性的LB平板上。将平板于37℃孵箱内培养16h。
重组质粒的鉴定：挑取单克隆菌落于反应管中，加入10μl ddH 2O重悬混匀，取1μl作为模板，以引物PAO A1-45和PAO B2-48进行菌落PCR。PCR结果通过琼脂糖凝胶电泳进行鉴定。取菌落PCR正确的单克隆菌落接种到5ml卡那霉素抗性的LB培养基中，37℃振荡培养12h。使用质粒提取试剂盒(Axygen公司)小量提取质粒DNA。取5μl质粒，限制性内切酶消化，通过琼脂糖凝胶电泳确认酶切结果，挑选酶切鉴定正确的重组质粒，选择相应的引物，送Tsingke公司进行DNA测序分析，确认目的基因序列阅读框的正确性。
(3)扩增获得含有两个凝血因子IIa切割位点的proPAO DNA片段
设计引物PAO C1-49上游和PAO C2-50下游，分别以引物PAO A1-45上游和PAO C2-50下游，PAO C1-49上游和PAO B2-48下游，以含有一个凝血因子IIa切割位点的质粒DNA为模板，分别扩增含有第二个凝血因子IIa切割位点(位于前导肽与α亚基之间)的两部分DNA片段C和D。PCR反应体系和条件同(1)。将使用引物PAO A1-45和PAO C2-50扩增得到的C片段与使用引物PAO C1-49和PAO B2-48得到的D片段进行重叠延伸PCR。引物使用PAO A1-45上游和PAO B2-48下游。PCR反应体系和条件同(1)。得到纯的含有两个凝血因子IIa切割位点的proPAO DNA片段，其对应的氨基酸序列见SEQ ID NO:2。
(4)克隆含有两个凝血因子IIa切割位点的proPAO DNA片段
步骤同(2)。
2.凝血因子底物特异性proPAO蛋白的表达与纯化
将前述含有两个凝血因子IIa切割位点的修饰的proPAO DNA片段的重组质粒转化入E.coli BL21(DE3)。挑取平板上的单克隆菌落，接种到5ml LB培养基中，37℃振荡培养12h。将上述全部培养物接种到含400ml新鲜LB培养基中，37℃振荡培养3h，当菌液OD 600nm达到0.6，向培养物中加入终浓度为0.5mM的IPTG，20℃继续培养16h。将培养物收集于50ml离心管中，4℃，6000rpm离心20min。弃上清液。
菌体溶解：将离心得到的菌体沉淀使用缓冲液A[50mM KH 2PO 4/K 2HPO 4，300mM NaCl，pH8.0]充分重悬。
菌体破碎：选择超声破碎。超声破碎条件为功率20％，超声2s间歇4s，5-30min。待菌液清亮后，于4℃，13000rpm离心30min，收集上清液。
镍柱亲和层析：向上清中加入0.8ml 50％的Ni-NTA悬液(QIAGEN公司)，于4℃摇床匀速混匀1h。将混合液加入层析柱中，上清在重力作用下流出，Ni-NTA基质沉淀于层析柱中。向层析柱中加入1倍柱体积的缓冲液A，洗涤层析柱，重复三次。待缓冲液A流净，向层析柱中加入5ml含有20mM咪唑的缓冲液A，洗脱杂质蛋白，重复三次。接着，向层析柱中分次加入2ml洗脱缓冲液B[50mM KH 2PO 4/K 2HPO 4，300mM NaCl，250mM Imidazole，pH8.0]，洗脱目的蛋白。收集蛋白洗脱液于1.5ml离心管中。
脱盐柱层析与保存：将得到的目的蛋白洗脱液于4℃，13000rpm离心30min，取上清液使用ATKA蛋白纯化仪(Amersham公司)进行脱盐层析，使蛋白处于缓冲液C[10mM KH 2PO 4/K 2HPO 4，pH8.0]中，加入甘油至10％，于-80℃保存备用。同时使用蛋白质定量试剂盒(Beyotime公司)对纯化产物进行含量测定，将目的蛋白样品进行SDS-PAGE电泳以检测蛋白的表达与纯化情况。电泳图如图1所示。
3.凝血因子IIa的酶解验证
取人的凝血因子IIa纯品(Sigma公司)，按反应体系(凝血因子IIa纯品1μg，修饰的proPAO 10μg，30mM KH 2PO 4/Na 2HPO 4，pH7.37)，37℃孵育3h后，SDS-PAGE验证水解效率。电泳图如图2所示。由图2可以看出凝血因子IIa可以成功地对修饰的proPAO进行切割。从左至右的每个泳道为未被切割的修饰的proPAO，凝血因子IIa将修饰的proPAO切割成具有较小迁移率的片段，商品化凝血因子IIa，蛋白marker。
4.凝血因子IIa活性检测方法的建立
选择Trinder显色反应，并优化相应的显色反应底物浓度、缓冲液、反应条件，检测条件，确定两种反应试剂R1、R2的组成及检测波长，初步建立检测体系。
反应试剂R1：TOOS(N-乙基-N-(2-羟基-3-磺丙基)-3-甲基苯胺钠盐)；反应试剂R2：L-Phe，HRP，4-AAT。吸光度检测反应体系：凝血因子IIa纯品(不同活力单位)，凝血因子IIa底物特异性proPAO，反应试剂R1，反应试剂R2。将上述组分加入酶标板小孔中，使用酶标仪振荡混匀反应体系，检测555nm和700nm处的吸光度随时间的变化。
数据处理与绘图：△Abs＝Abs 555nm-Abs 700nm，以时间t为横坐标，△Abs为纵坐标，绘制不同活力单位的FIIa(Sigma公司)纯品酶解动力学的散点图。检测结果如图3所示。由图3可以看出随着反应体系中凝血因子IIa活力单位的增加，修饰的proPAO被切割的反应速率随之增大。
分别对各散点图进行函数拟合以描述相应的反应速率，使用简单线性回归检验动力学方程参数与凝血因子FIIa活力单位间的关系。结果如图4所示。由图4可以看出得到的动力学方程参数与FIIa活力单位间成线性相关，R 2＝0.9989，解释度良好。
优化检测反应条件：分别配制含不同底物浓度的检测试剂。控制其他底物及反应条件不变，选择含有同一底物的不同浓度的试剂，以经凝血因子IIa纯品水解后的特异底物作为样品进行反应。使用酶标仪连续监测反应体系的吸光度变化，以时间t为横坐标，△Abs为纵坐标，绘制散点图。确定的最优反应底物浓度分别为8mM TOOS，7.5mM L-phenylalanine，6mM 4-AAT，5U/ml HRP和0.20μg/μl经修饰proPAO，最佳反应温度为37℃。
优化检测反应pH值：在凝血因子IIa活性的最适pH条件和血液pH条件下，配制梯度pH的磷酸盐缓冲液。以经凝血因子IIa纯品水解后的特异底物作为样品进行反应，使用酶标仪连续监测反应体系的吸光度变化。以时间t为横坐标，△Abs为纵坐标，绘制散点图。最佳pH值为7.35-7.45。
5.临床样本的初步检测
(1)枸橼酸钠抗凝血浆标本的检测实验
收集样本：选取2017年6月至9月期间北京医院普通门诊(非心血管疾病、非妊娠、非骨科住院复查患者、非甲状腺疾病等有高血栓风险的患者，非抗凝治疗及避孕药使用的患者)的枸橼酸钠抗凝血浆样本，枸橼酸钠：血液＝1:9(枸橼酸钠终浓度约为0.109mmol/L)。为最大程度的避免随机误差，将血浆标本以10份为一组混匀制备血浆盘。以混合血浆为样本进行检测。
样品检测：在酶标板中加入下列成分(混合血浆样本，反应试剂R1(90μl)，反应试剂R2(90μl)和特异底物proPAO(10μl))后，设置酶标仪的反应程序：振荡混匀，检测555nm和700nm处吸光度随时间的变化。
(2)血浆激活实验
在枸橼酸钠抗凝血浆标本的检测反应体系(血浆样本，R1，R2和修饰的proPAO)中加入CaCl 2，同时加入组织因子(TF)，以充分激活待测血浆中的凝血因子IIa。
(3)链激酶实验
样品准备：备枸橼酸钠抗凝的混合血浆标本，使用20μl链激酶+100μl混合血浆标本制备预处理的血浆样本。对照样本使用20μl ddH 2O+100μl混合血浆标本制成。在干净的酶标板中加入下列成分(预处理样本/对照样本(20μl)，反应试剂R1(90μl)，反应试剂R2(90μl)和修饰的proPAO(10μl))后，使用酶标仪进行反应检测。检测结果如图5所示(图注方框中物质预先混合)。 由图5可以看出使用链激酶预处理血浆后，激活状态的凝血因子IIa引起了酶联激活反应。
(4)凝血酶与吸光度之间定量关系的建立
Trinder反应是较为成熟的临床生化检验常用反应，其反应条件、底物及过氧化物酶的浓度的选择均根据过氧化物酶的反应参数而得。为保证信号检测模块的反应速度只与过氧化氢浓度成正比，其余底物及酶在反应体系中的浓度均远高于K m值。
对信号检测的吸光度值与待测样本凝血因子IIa的浓度间的数量关系进行分析。在一级反应阶段时，信号检测的吸光度值与待测样本凝血因子IIa的浓度间成线性关系。根据相应的苯丙氨酸氧化酶酶原激活的时间反应曲线计算，得到吸光度值变化(△Abs)与凝血因子IIa标准品的浓度(c (FIIa))之间的方程式y＝ax+b，将实际测得的吸光度值变化代入上述方程式可以计算得到待测样本中凝血因子IIa的浓度。
实施例2.凝血因子Xa活性检测
由于凝血因子Xa活性检测多了一步中间蛋白酶原的激活反应，因此，首先合成了修饰的proNisP(Streptococcus lactis)酶原DNA片段，该DNA片段的前导肽序列与催化亚基序列之间包含有凝血因子Xa的特异识别序列IEGR(SEQ ID NO:17)的DNA序列。完整氨基酸序列图见SEQ ID NO:3。对此DNA片段进行限制性内切酶消化(BamHI，NcoI)，与经过同样的两个限制性内切酶酶切回收得到的质粒载体pRSFDuet-1相连。将连接产物进行转化并鉴定得到正确序列的重组质粒，将重组质粒转化E.coli BL21(DE3)。挑取平板上的单克隆菌落，进行修饰蛋白proNisP的诱导表达，镍柱亲和层析和脱盐层析，表达与纯化的步骤参见凝血因子IIa底物特异性proPAO的表达与纯化。将目的蛋白样品进行SDS-PAGE电泳以检测蛋白的表达与纯化情况。电泳如图6所示。
接着，以编码SEQ ID NO:1的proPAO基因为模板，运用三对相应引物进行重叠延伸PCR，最终获得含有二个NisP切割位点的DNA片段，完成Xa因子酶联检测特异底物序列的构建，其编码的氨基酸序列图见SEQ ID NO:4。修饰的proPAO特异底物蛋白的表达与纯化参见凝血因子IIa实施方 案中的相应描述。目的蛋白的SDS-PAGE电泳如图6所示。
凝血因子Xa的酶解验证：取牛的凝血因子Xa纯品(NEB公司)，按反应体系(Xa因子纯品0.5μg，修饰的proNisP 2μg，修饰的proPAO 6μg，30mM KH 2PO 4/Na 2HPO 4，pH 7.37)，37℃孵育2.5h后，SDS-PAGE验证水解效率。电泳图如图7所示。由图7可以看出凝血因子Xa可以成功地通过切割修饰proNisP实现对修饰proPAO的切割。从左至右的每个泳道为蛋白marker，未被切割的修饰proNisP，凝血因子Xa对修饰proNisP的切割，凝血因子Xa通过切割修饰proNisP进行对修饰proPAO的切割，未被切割的修饰proPAO，未被切割的修饰proNisP和修饰proPAO，蛋白marker。
Xa因子活性检测方法的建立：参见凝血因子IIa活性检测方法，反应体系包含Xa因子纯品或枸橼酸钠抗凝血浆样本(20μl)，修饰的proNisP(6μl)，修饰的proPAO(20μl)，反应试剂R1(90μl)，反应试剂R2(90μl)。将上述组分加入酶标板小孔中，使用酶标仪振荡混匀反应体系，检测555nm和700nm处的吸光度随时间的变化。
数据处理与绘图：△Abs＝Abs 555nm-Abs 700nm，以时间t为横坐标，△Abs为纵坐标，绘制酶解动力学的散点图，分别对各散点图进行函数拟合以描述相应的反应速率。枸橼酸钠抗凝血浆样本的处理与激活方法参见凝血因子IIa活性检测的实施方案。检测结果如图8所示(图注方框中物质预先混合)。由图8可以看出使用链激酶预处理血浆后，激活状态的凝血因子Xa引起了酶联激活反应。
实施例3.纤溶酶Plasmin活性检测
以编码SEQ ID NO:1的proPAO基因为模板，运用三对相应引物进行重叠延伸PCR，最终获得含有二个Plasmin切割位点(GYRA，SEQ ID NO:23)的DNA片段，完成Plasmin活性检测特异底物序列的构建。氨基酸序列图见SEQ ID NO:5。修饰proPAO特异底物蛋白的表达与纯化参见凝血因子IIa实施方案中的相应描述。
Plasmin活性检测方法的建立：反应体系包含枸橼酸钠抗凝血浆样本(20μl)，特异底物修饰的proPAO(5μl)，反应试剂R1(90μl)，反应试剂R2(90μl)。将上述组分加入酶标板小孔中，使用酶标仪振荡混匀反应体系，检测555nm 和700nm处的吸光度随时间的变化。数据处理与绘图参见凝血因子IIa活性检测的实施方案。由于链激酶可以直接激活血浆中的纤溶酶原使之生成纤溶酶，因此，血浆中全激活状态的Plasmin的活性可以通过链激酶的加入直接被检测。检测结果如图9所示。由图9可以看出血浆中的Plasminogen被链激酶激活后生成Plasmin，后者引起酶联激活反应。
实施例4.Caspase-3活性检测
以编码SEQ ID NO:1的proPAO基因为模板，运用三对相应引物进行重叠延伸PCR，最终获得含有二个Caspase-3切割位点的DNA片段，完成Caspase-3活性检测特异底物序列的构建。氨基酸序列图见SEQ ID NO:6。修饰proPAO特异底物蛋白的表达与纯化参见凝血因子IIa实施方案中的相应描述。
Caspase-3蛋白的活性检测方法如下：将定量的Caspase-3与不同浓度的显色底物Ac-DEVD-pNA加入酶标板小孔中，反应体系包含Caspase-3(2μl)，Ac-DEVD-pNA(2μl)，30mM KH 2PO 4/Na 2HPO 4，pH8.0(200μl)，使用酶标仪振荡混匀反应体系，检测405nm处的吸光度随时间的变化。检测结果如图10所示。由图10可以看出在一定浓度的Caspase-3的条件下，提高底物Ac-DEVD-pNA的浓度可以增大反应速率。该反应可以得到一定质量Caspase-3对应的酶活力单位。
Caspase-3活性检测方法的建立：反应体系包含血清样本(20μl)，特异底物修饰proPAO(5μl)，反应试剂R1(90μl)，反应试剂R2(90μl)。将上述组分加入酶标板小孔中，使用酶标仪振荡混匀反应体系，检测555nm和700nm处的吸光度随时间的变化。数据处理与绘图参见凝血因子IIa活性检测的实施方案。该反应可以直接检测血清中Caspase-3对含有Caspase-3特异切割序列的修饰proPAO的激活反应。检测结果如图11所示。由图11可以看出不同血清标本中所含的Caspase-3对修饰proPAO的酶联激活反应速率不同。
实施例5.C5转化酶活性检测
以编码SEQ ID NO:1的proPAO基因为模板，运用三对相应引物进行重叠延伸PCR，最终获得含有二个C5转化酶切割位点的DNA片段，完成C5 转化酶活性检测特异底物序列的构建。氨基酸序列图见SEQ ID NO:7。C5转化酶特异底物蛋白修饰proPAO的表达与纯化参见凝血因子IIa实施方案中的相应描述。
C5转化酶活性检测方法的建立：反应体系包含血清样本(20μl)，特异底物修饰proPAO(15μl)，反应试剂R1(90μl)，反应试剂R2(90μl)。将上述组分加入酶标板小孔中，使用酶标仪振荡混匀反应体系，检测555nm和700nm处的吸光度随时间的变化。数据处理与绘图参见凝血因子IIa活性检测的实施方案。该反应可以直接检测血清中C5转化酶对含有该酶特异切割序列的修饰proPAO的激活反应。检测结果如图12所示。由图12可以看出不同血清标本中所含的C5转化酶对修饰proPAO的酶联激活反应速率不同。
比较例6
除了苯丙氨酸氧化酶酶原，本发明还选用了其他的酶原并进行特异底物的改造，用来检测人体内蛋白酶的活性。在一些实施方案中，选用了两种不同来源的多酚氧化酶酶原，分别为Prophenoloxidase(signal crayfish Pacifastacus leniusculus)(简称为proCPO)和Prophenoloxidase 1(Drosophila melanogaster)(简称为proDPO)。检测结果显示，相应蛋白酶对修饰底物proCPO的激活作用在大约100min后才开始显现，相应蛋白酶对修饰底物proDPO几乎无激活作用(如图13所示)。该结果表明这两种重组酶原底物的灵敏度较低，检测时间较长，并且在血浆中不稳定。
这些比较实验显示了苯丙氨酸氧化酶酶原作为底物酶的优势，它具有特异性好，灵敏度高，检测时间短，方便制备等多种优点。
比较例7
关于苯丙氨酸氧化酶酶原的改造位置和具体序列，本发明中还进行了将特定酶切位点序列只插入到α亚基与β亚基之间，试验过程如下：以编码SEQ ID NO:1的proPAO基因为模板，运用二对相应引物进行重叠延伸PCR，获得含有一个凝血因子IIa切割位点的DNA片段(苯丙氨酸氧化酶酶原序列中的α亚基与β亚基间)，完成凝血因子IIa活性检测特异底物序列的构建。修饰proPAO的表达与纯化、凝血因子IIa的活性检测方法参见凝血因子IIa实 施方案中的相应描述。检测结果如图14所示。由图14可以看出血浆样本中凝血因子IIa的活性几乎不能被检测到。
可以看出，苯丙氨酸氧化酶酶原的前导肽序列和α亚基之间、α亚基与β亚基之间分别含有特定的酶切位点时，才能够完成某种蛋白酶活性的检测。
比较例8
对于某种特定的蛋白酶激活反应，在苯丙氨酸氧化酶酶原中没有插入该种蛋白酶偏爱的切割位点序列时，无法检测酶原激活反应。试验过程如下：以proPAO基因为模板，运用三对相应引物进行重叠延伸PCR，获得含有一个来源于金黄色葡萄球菌(Staphylococcus aureus)的SplB蛋白酶切割位点(WELQ，SEQ ID NO:68)的DNA片段，完成特异底物序列的构建。特异底物蛋白修饰proPAO的表达与纯化、凝血因子IIa的活性检测方法参见凝血因子IIa实施方案中的相应描述。检测结果如图15所示。由图15可以看出使用不含有凝血因子IIa切割位点序列的proPAO时，无法检测到血浆样本中凝血因子IIa的活性，同时无显色反应。因此在本实施案例中，选择在苯丙氨酸氧化酶酶原中插入相应蛋白酶特异切割序列，使得检测反应具有速度快、灵敏度高等优势。
序列表
SEQ ID NO:1：来自Psuedomonas Sp.P-501的苯丙氨酸氧化酶酶原氨基酸序列
Figure PCTCN2020083317-appb-000003
前导肽序列以粗体双下划线显示，α亚基以虚线下划线显示，β亚基以实线下划线显示，连接α与β亚基的前体接头以粗体显示。
SEQ ID NO:2：含有两个凝血因子IIa切割位点的来自Psuedomonas Sp.P-501的苯丙氨酸氧化酶酶原的氨基酸序列(凝血因子IIa底物特异性proPAO)
Figure PCTCN2020083317-appb-000004
前导肽序列以粗体双下划线显示，α亚基以虚线下划线显示，β亚基以 实线下划线显示。凝血因子IIa的切割识别序列LRPR分别位于前导肽序列与α亚基之间，α亚基与β亚基之间，以粗斜体显示。
SEQ ID NO:3：含有两个凝血因子Xa切割位点的来自乳酸链球菌(Streptococcus lactis)的NisP酶酶原proNisP(凝血因子Xa底物特异性proNisP)
Figure PCTCN2020083317-appb-000005
前导肽序列以双下划线显示。凝血因子Xa的切割识别序列IEGR分别位于前导肽序列与催化亚基之间，以粗斜体显示。
SEQ ID NO:4：含有两个NisP切割位点的来自Psuedomonas Sp.P-501的苯丙氨酸氧化酶酶原(NisP底物特异性proPAO)
Figure PCTCN2020083317-appb-000006
前导肽序列以粗体双下划线显示，α亚基以虚线下划线显示，β亚基以实线下划线显示。NisP的识别切割序列ASPRI分别位于前导肽序列与α亚基之间，α亚基与β亚基之间，以粗斜体显示。
SEQ ID NO:5：含有两个Plasmin切割位点的来自Psuedomonas Sp.P-501的苯丙氨酸氧化酶酶原(Plasmin特异性proPAO)
Figure PCTCN2020083317-appb-000007
前导肽序列以粗体双下划线显示，α亚基以虚线下划线显示，β亚基以实线下划线显示。Plasmin的识别切割序列GYRA分别位于前导肽序列与α 亚基之间，α亚基与β亚基之间，以粗斜体显示。
SEQ ID NO:6：含有两个Caspase-3切割位点的来自Psuedomonas Sp.P-501的苯丙氨酸氧化酶酶原(Caspase-3底物特异性proPAO)
Figure PCTCN2020083317-appb-000008
前导肽序列以粗体双下划线显示，α亚基以虚线下划线显示，β亚基以 实线下划线显示。Caspase-3的识别切割序列DEVD分别位于前导肽序列与α亚基之间，α亚基与β亚基之间，以粗斜体显示。
SEQ ID NO:7：含有两个C5转化酶切割位点的来自Psuedomonas Sp.P-501的苯丙氨酸氧化酶酶原(C5转化酶底物特异性proPAO)
Figure PCTCN2020083317-appb-000009
前导肽序列以粗体双下划线显示，α亚基以虚线下划线显示，β亚基以实线下划线显示。C5转化酶的识别切割序列QLGRLHMK分别位于前导肽序列与α亚基之间，α亚基与β亚基之间，以粗斜体显示。
SEQ ID NO:8：凝血因子IIa的特异性切割序列
Figure PCTCN2020083317-appb-000010
其中“Xaa”为任意氨基酸残基，“Xab”为优选自Pro、Ala、Gly或Val的氨基酸残基，“Xac”为优选自Ser、Ala、Gly的氨基酸残基
SEQ ID NO:9：凝血因子IIa的特异性切割序列
Figure PCTCN2020083317-appb-000011
SEQ ID NO:10：凝血因子IIa的特异性切割序列
Figure PCTCN2020083317-appb-000012
SEQ ID NO:11：凝血因子IIa的特异性切割序列
Figure PCTCN2020083317-appb-000013
SEQ ID NO:12：凝血因子IIa的特异性切割序列
Figure PCTCN2020083317-appb-000014
SEQ ID NO:13：凝血因子VIIa的特异性切割序列
Figure PCTCN2020083317-appb-000015
SEQ ID NO:14：凝血因子IXa的特异性切割序列
Figure PCTCN2020083317-appb-000016
其中“Xaa”为任意氨基酸残基
SEQ ID NO:15：凝血因子IXa的特异性切割序列
Figure PCTCN2020083317-appb-000017
SEQ ID NO:16：凝血因子Xa的特异性切割序列
Figure PCTCN2020083317-appb-000018
其中“Xaa”为任意氨基酸残基，“Xad”为优选自Ala或Ile的氨基酸残基，“Xae”为优选自Pro、Phe或Gly的氨基酸残基，“Xaf”为选自Arg或Lys的氨基酸残基
SEQ ID NO:17：凝血因子Xa的特异性切割序列
Figure PCTCN2020083317-appb-000019
SEQ ID NO:18：凝血因子Xa的特异性切割序列
Figure PCTCN2020083317-appb-000020
SEQ ID NO:19：凝血因子XIa的特异性切割序列
Figure PCTCN2020083317-appb-000021
其中“Xag”为优选自Lys或Asp的氨基酸残基，“Xah”为优选自Phe或Leu的氨基酸残基
SEQ ID NO:20：凝血因子XIa的特异性切割序列
Figure PCTCN2020083317-appb-000022
SEQ ID NO:21：凝血因子XIIa的特异性切割序列
Figure PCTCN2020083317-appb-000023
SEQ ID NO:22：纤溶酶Plasmin的特异性切割序列
Figure PCTCN2020083317-appb-000024
其中“Xaa”为任意氨基酸残基，“Xai”为优选自Arg或Lys的氨基酸残基，“Xaj”为优选自Ala、Ser、Gly或Arg的氨基酸残基
SEQ ID NO:23：纤溶酶Plasmin的特异性切割序列
Figure PCTCN2020083317-appb-000025
SEQ ID NO:24：纤溶酶Plasmin的特异性切割序列
Figure PCTCN2020083317-appb-000026
SEQ ID NO:25：Caspase-2的特异性切割序列
Figure PCTCN2020083317-appb-000027
其中“Xaa”为任意氨基酸残基
SEQ ID NO:26：Caspase-3的特异性切割序列
Figure PCTCN2020083317-appb-000028
其中“Xak”为优选自Asp或Glu的氨基酸残基
SEQ ID NO:27：Caspase-3的特异性切割序列
Figure PCTCN2020083317-appb-000029
SEQ ID NO:28：Caspase-6的特异性切割序列
Figure PCTCN2020083317-appb-000030
其中“Xaa”为任意氨基酸残基
SEQ ID NO:29：Caspase-7的特异性切割序列
Figure PCTCN2020083317-appb-000031
其中“Xaa”为任意氨基酸残基
SEQ ID NO:30：Caspase-8的特异性切割序列
Figure PCTCN2020083317-appb-000032
其中“Xal”为优选自Asp或Leu的氨基酸残基，“Xam”为优选自Glu或Ser的氨基酸残基
SEQ ID NO:31：Caspase-8的特异性切割序列
Figure PCTCN2020083317-appb-000033
SEQ ID NO:32：Caspase-9的特异性切割序列
Figure PCTCN2020083317-appb-000034
其中“Xaa”为任意氨基酸残基，“Xan”为优选自Asp或Glu的氨基酸残基
SEQ ID NO:33：Caspase-10的特异性切割序列
Figure PCTCN2020083317-appb-000035
其中“Xaa”为任意氨基酸残基，“Xao”为优选自Glu、Gln或Ser的氨基酸残基，“Xap”为优选自Thr或Val的氨基酸残基
SEQ ID NO:34：Caspase-14的特异性切割序列
Figure PCTCN2020083317-appb-000036
其中“Xaa”为任意氨基酸残基
SEQ ID NO:35：补体途径C3/C5转化酶的特异性切割序列
Figure PCTCN2020083317-appb-000037
SEQ ID NO:36：补体途径C3/C5转化酶的特异性切割序列
Figure PCTCN2020083317-appb-000038
SEQ ID NO:37：MMP-8的特异性切割序列
Figure PCTCN2020083317-appb-000039
其中“Xaa”为任意氨基酸残基，“Xaq”为优选自Pro、Ala或Ser的氨基酸残基
SEQ ID NO:38：MMP-11的特异性切割序列
Figure PCTCN2020083317-appb-000040
其中“Xaa”为任意氨基酸残基
SEQ ID NO:39：MMP-12的特异性切割序列
Figure PCTCN2020083317-appb-000041
其中“Xar”为优选自Pro、Ala或Gly的氨基酸残基,“Xas”为优选自Ala或Gly的氨基酸残基
SEQ ID NO:40：MMP-13的特异性切割序列
Figure PCTCN2020083317-appb-000042
其中“Xaa”为任意氨基酸残基，“Xat”为优选自Leu、Ile或Val的氨基酸残基
SEQ ID NO:41：MMP-13的特异性切割序列
Figure PCTCN2020083317-appb-000043
SEQ ID NO:42：MMP-20的特异性切割序列
Figure PCTCN2020083317-appb-000044
其中“Xaa”为任意氨基酸残基，“Xau”为优选自Leu或Met的氨基酸残基
SEQ ID NO:43：MMP-20的特异性切割序列
Figure PCTCN2020083317-appb-000045
SEQ ID NO:44：MMP-20的特异性切割序列
Figure PCTCN2020083317-appb-000046
SEQ ID NO:45：上游PAO A1-45
5′-catgccatggtgggcgttaccgtcattccccggctgc-3′
SEQ ID NO:46：下游PAO A2-46
5′-gcccgcgtaccttgatcgcacgtggacgcagtcccgggcggtcatgaaga-3′
SEQ ID NO:47：上游PAO B1-47
5′-tcttcatgaccgcccgggactgcgtccacgtgcgatcaaggtacgcgggc-3′
SEQ ID NO:48：下游PAO B2-48
5′-ggaattccatatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′
SEQ ID NO:49：上游PAO C1-49
5′-cggcctgaaggacgagaagctgcgtccacgtaagattgccaccaccgttg-3′
SEQ ID NO:50：下游PAO C2-50
5′-caacggtggtggcaatcttacgtggacgcagcttctcgtccttcaggccg-3′
SEQ ID NO:51：下游PAO A2-51
5′-gcccgcgtaccttgatcgcaatacgaggagatgctcccgggcggtcatga-3′
SEQ ID NO:52：上游PAO B1-52
5′-tcatgaccgcccgggagcatctcctcgtattgcgatcaaggtacgcgggc-3′
SEQ ID NO:53：上游PAO C1-53
5′-cctgaaggacgagaaggcatctcctcgtattaagattgccaccaccgttg-3′
SEQ ID NO:54：下游PAO C2-54
5′-caacggtggtggcaatcttaatacgaggagatgccttctcgtccttcagg-3′
SEQ ID NO:55：下游PAO A2-55
5′-cagcccgcgtaccttgatcgcacgataacctcccgggcggtcatgaagaa-3′
SEQ ID NO:56：上游PAO B1-56
5′-ttcttcatgaccgcccgggaggttatcgtgcgatcaaggtacgcgggctg-3′
SEQ ID NO:57：上游PAO C1-57
5′-cggcctgaaggacgagaagggttatcgtgcaaagattgccaccaccgttg-3′
SEQ ID NO:58：下游PAO C2-58
5′-caacggtggtggcaatctttgcacgataacccttctcgtccttcaggccg-3′
SEQ ID NO:59：下游PAO A2-59
5′-gcccgcgtaccttgatcgcatcaacttcgtctcccgggcggtcatgaaga-3′
SEQ ID NO:60：上游PAO B1-60
5′-tcttcatgaccgcccgggagacgaagttgatgcgatcaaggtacgcgggc-3′
SEQ ID NO:61：上游PAO C1-61
5′-cggcctgaaggacgagaaggacgaagttgataagattgccaccaccgttg-3′
SEQ ID NO:62：下游PAO C2-62
5′-caacggtggtggcaatcttatcaacttcgtccttctcgtccttcaggccg-3′
SEQ ID NO:63：下游PAO A2-63
5′-cgcgtaccttgatcgctttcatgtgcagacgacccagttgtcccgggcggtcatga-3′
SEQ ID NO:64：上游PAO B1-64
5′-tcatgaccgcccgggacaactgggtcgtctgcacatgaaagcgatcaaggtacgcg-3′
SEQ ID NO:65：上游PAO C1-65
5′-cctgaaggacgagaagcaactgggtcgtctgcacatgaagattgccaccaccgttg-3′
SEQ ID NO:66：下游PAO C2-66
5′-caacggtggtggcaatcttcatgtgcagacgacccagttgcttctcgtccttcagg-3′
SEQ ID NO:67：NisP的特异识别序列
Figure PCTCN2020083317-appb-000047
SEQ ID NO:68：SplB蛋白酶切割位点
Figure PCTCN2020083317-appb-000048

Claims (13)

  1. 一种经修饰的苯丙氨酸氧化酶酶原，其特征在于：所述修饰是在苯丙氨酸氧化酶酶原序列中的前导序列与α亚基间以及α亚基与β亚基间引入蛋白酶的特异性识别位点序列。
  2. 如权利要求1所述的经修饰的苯丙氨酸氧化酶酶原，其来源于Pseudomonas Sp.P-501，东湖假单胞菌(Pseudomonas donghuensis)，茄科雷尔氏菌(Ralstonia solanacearum)UW551或洋葱伯克氏菌(Burkholderia cepacia)ATCC 25416。
  3. 如权利要求1或2所述的经修饰的苯丙氨酸氧化酶酶原，所述蛋白酶选自凝血级联蛋白酶，例如凝血因子IIa、Va、VIIa、VIIIa、IXa、Xa、XIa或XIIa；纤溶酶；含半胱氨酸的天冬氨酸蛋白酶，例如Caspase-1、Caspase-2、Caspase-3、Caspase-4、Caspase-5、Caspase-6、Caspase-7、Caspase-8、Caspase-9、Caspase-10或Caspase-11；补体途径蛋白酶，例如因子C1、C2、C3、C4、C5、C6、C7、C8、C9或C3/C5转换酶；MMP家族蛋白酶；NisP酶。
  4. 如权利要求1或2所述的经修饰的苯丙氨酸氧化酶酶原，所述蛋白酶的特异识别位点序列如SEQ ID NO:8-44、67任一所示。
  5. 如权利要求1-4任一项所述的经修饰的苯丙氨酸氧化酶酶原，所述苯丙氨酸氧化酶酶原序列如SEQ ID NO:1所示。
  6. 如权利要求1-5任一项所述的经修饰的苯丙氨酸氧化酶酶原，其序列如SEQ ID NO:2、4-7任一项所示。
  7. 权利要求1-6中任一项所述的经修饰的苯丙氨酸氧化酶酶原用于检测样品中蛋白酶活性的用途。
  8. 如权利要求7所述的用途，所述样品选自体液、全血、血浆、血清或组织。
  9. 一种分离的核酸，其编码权利要求1-6中的任一项所述的经修饰的苯丙氨酸氧化酶酶原。
  10. 一种表达载体，其包含权利要求9所述的分离的核酸。
  11. 用权利要求10所述的表达载体转化或转染的宿主细胞。
  12. 一种检测样品中蛋白酶活性的试剂盒，其包括权利要求1-6任一项所述的经修饰的苯丙氨酸氧化酶酶原。
  13. 如权利要求12所述的试剂盒，所述试剂盒还包含经修饰的NisP蛋白酶酶原，其在前导肽和催化亚基之间包含酶特异性识别位点序列，例如凝血因子Xa的特异识别序列。
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