CN113563484A - Fusion protein with G11-scFv-Nluc dual-functional activity and application thereof - Google Patents
Fusion protein with G11-scFv-Nluc dual-functional activity and application thereof Download PDFInfo
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Abstract
The invention discloses a fusion protein with G11-scFv-Nluc dual-functional activity and application thereof, aiming at providing a fusion protein with better specificity and signal amplification effect, and establishing a hypersensitive bioluminescence immunoassay kit by adopting the fusion protein, which is used for detecting the content of glycocholic acid in urine and has good application value and prospect; the G11-scFv-Nluc fusion protein is prepared by the following method: the method is characterized in that nano-luciferase is used as a bioluminescent catalyst, an anti-glycocholic acid single-chain antibody (G11-scFv) and the nano-luciferase are fused and cloned into a pET22b expression vector, and the method belongs to the technical field of immunodetection.
Description
Technical Field
The invention belongs to the technical field of immunodetection, relates to a fusion protein, in particular to a fusion protein with G11-scFv-Nluc bifunctional activity, and also relates to an application of the fusion protein with G11-scFv-Nluc bifunctional activity.
Background
Glycocholic acid is one of combined cholic acids formed by combining cholic acid and glycine, is a product of cholesterol metabolism in liver cells, has sensitivity in liver disease diagnosis in content determination, and is an important index for evaluating liver functions. Glycocholic acid is used as a biomarker for prompting the diseases of the glycocholic acid, and the immunoassay detection of the small molecules provides important basis for diagnosis, treatment and prognosis analysis of liver diseases. Related researches indicate that bile acid is obviously excreted through the kidney and is related to the degree of liver and gall diseases, so that the detection of glycocholic acid in urine has certain clinical guiding value. Clinically, the glycocholic acid is detected by taking venous blood, and the glycocholic acid in urine of some children patients and patients who are inconvenient to take venous blood has convenience and guiding significance for disease diagnosis.
In immunoassay methods, it is a constant pursuit to enhance sensitivity. In immunoassay methods, it is a constant pursuit to enhance sensitivity. In order to improve the sensitivity of the detection method, the traditional ELISA is usually combined with biotin and avidin, fluorescence, chemiluminescence and other modification methods. The luciferase labeling technology is a novel biological reaction amplification system and can be organically combined with ELISA. The luciferase has the advantages of high sensitivity and specificity, simple operation, rapid reaction and the like, and is applied to scientific research such as rapid detection and the like at present. However, the conventional luciferase has disadvantages in use, such as susceptibility of fluorescence intensity to factors such as pH, ionic strength and temperature, dependence on ATP, etc., and thus prevents further use. In recent years, the defect of the traditional luciferase is well compensated by the appearance of a novel nano-luciferase.
In order to improve the stability and operability of the detection method, the patent develops an immunoassay method based on the nano-luciferase, and the nano-luciferase has the advantages of small molecular weight, no need of ATP, high luminous efficiency, relatively stable physicochemical properties at various pHs and temperatures and the like, and can be widely used in immunoassay methods.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a G11-scFv-Nluc fusion protein which has better specificity and signal amplification effect.
The invention also aims to provide application of the G11-scFv-Nluc fusion protein in detecting the content of glycocholic acid in urine.
In order to solve the above technical problems, the present invention provides the following technical solutions:
a G11-scFv-Nluc bifunctional fusion protein, wherein the G11-scFv-Nluc fusion protein is prepared by the following steps:
the nano-luciferase is used as a bioluminescent catalyst, and an anti-glycocholic acid single-chain antibody (G11-scFv) is fused with the nano-luciferase and cloned into a pET22b expression vector to obtain the antibody.
Further, the G11-scFv-Nluc bifunctional fusion protein is prepared by the following steps:
(1) construction of G11-scFv-Nluc expression vector: cloning G11-scFv and Nluc genes by a PCR technology, splicing to obtain a fusion gene G11-scFv-Nluc, and obtaining an expression vector pET22b (+) -G11-scFv-Nluc by double enzyme digestion, gene recovery and enzyme connection;
(2) preparation of G11-scFv-Nluc fusion protein: the expression vector is transformed into a competent cell BL21 by a chemical transformation method, then the positive monoclonal bacteria are cultured, and the fusion protein is induced and expressed by an inducer.
Further, the construction of the G11-scFv-Nluc expression vector of the G11-scFv-Nluc bifunctional fusion protein in the step (1) specifically comprises the following steps:
cloning G11-scFv and Nluc genes respectively by taking SNF1, SNR1, SNF2 and SNR2 as primers;
performing overlap extension PCR technology by respectively taking the first round products G11-scFv and Nluc as templates and taking SNF1 and SNR2 as primers to obtain G11-scFv-Nluc;
and carrying out XholI and EcoRI double enzyme digestion on the pET22b vector and the G11scFv-Nluc gene respectively by using a second round of PCR, recovering the enzyme digestion product by cutting glue, and carrying out enzyme ligation again to obtain an expression vector pET22b (+) -G11-scFv-Nluc.
Further, the G11-scFv-Nluc fusion protein prepared in the step (2) of the G11-scFv-Nluc bifunctional fusion protein specifically comprises the following steps:
based on the expression vector pET22b (+) -G11-scFv-Nluc in the step (1), the whole expression vector is transferred into 100uLBL2 competent cells, ice bath is carried out for 25min, water bath heat shock is carried out for 90s at 42 ℃, and then the cells are quickly placed into an ice box for 2 min; adding 700uLLB culture medium, shaking at 37 ℃ for 1 hour, sucking a proper volume, and uniformly spreading the volume to a solid culture medium containing antibiotics for overnight culture; the next day, monoclonal bacteria are selected for culture, samples are sent for sequencing, a sequencing primer is T7, the monoclonal bacteria with positive sequencing accuracy are subjected to amplification culture, IPTG inducer is added for induction expression, B-per bacterial lysate is used for extracting protein, target protein is purified through a nickel affinity chromatography column, the protein is concentrated through ultrafiltration, and finally SDS-PAGE electrophoresis is adopted for determining the molecular weight and the purity of the protein.
The second technical scheme provided by the invention is the application of the G11-scFv-Nluc bifunctional active fusion protein as a glycocholic acid detection antibody in urine.
The third technical scheme provided by the invention is the G11-scFv-Nluc bifunctional active fusion protein, GCA-OVA and glycocholic acid.
Compared with the prior art, the invention has the beneficial effects that:
1) according to the technical scheme provided by the invention, nano luciferase is used as a bioluminescent catalyst, an anti-glycocholic acid single-chain antibody (G11-scFv) and the nano luciferase are fused and cloned into a pET22b expression vector, and the fusion protein with the antibody-enzyme (G11-scFv-Nluc) dual-function activity is obtained. The kit is high in specificity and good in sensitivity, the lowest detection limit is 0.131ug/mL, the sensitivity is improved by 38 times compared with a common enzyme-linked immunoassay method (5.08ug/mL), and an accurate, reliable and ultrahigh-sensitivity detection method is provided for rapid detection of glycocholic acid in urine;
2) the G11-scFv-Nluc fusion protein provided by the invention has better specificity and signal amplification effect, and the G11-scFv-Nluc fusion protein is adopted to establish a hypersensitive bioluminescence immunoassay kit for detecting the content of glycocholic acid in urine, so that the G11-scFv-Nluc fusion protein has good application value and prospect.
Drawings
FIG. 1 shows the DNA sequence and restriction enzyme sites of the PCR amplification primer of the present invention.
FIG. 2 is an SDS-PAGE electrophoretogram of the fusion protein (G11-scFv-Nluc) according to the present invention.
FIG. 3 is a competitive inhibition curve for glycocholic acid using the established bioluminescent immunoassay method of the present invention as described herein.
Detailed description of the invention
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1 construction of G11-scFv-Nluc expression vector
The G11-scFv gene is amplified by taking pComb3x-G11 as a template and SNF1 and SNR1 as primers, and the sequence is shown as SEQ ID NO. 1. The nanometer luciferase Nluc gene is obtained according to the accession Genbank, the gene sequence is from the Genbank JQ437370.1, and the nanometer luciferase Nluc report gene is 100-615 bp. The Nluc gene sequence is amplified by PCR technology by taking SNF2 and SNR2 as primers, and is shown as SEQ ID NO. 2.
Performing overlap extension PCR technology by respectively taking first round products G11-scFv and Nluc as templates and SNF1 and SNR2 as primers, splicing G11-scFv and Nluc genes, wherein the sequence of a linker gene between the G11-scFv and the Nluc genes is shown in figure 1; 6 His tags of histidine are added at the N end, and enzyme cutting sites EcoRI and XholI are inserted at both ends of the gene.
Carrying out double enzyme digestion on a pET22b (+) vector and a G11scFv-Nluc gene by Xhol I and EcoR I respectively, carrying out electrophoresis on enzyme digestion products by 0.7 percent and 1 percent agarose gel respectively, and recovering and purifying a target fragment by utilizing an omagelextraction kit; the recovered product was subjected to enzyme ligation overnight by using T4DNALigase, 16 ℃ PCR instrument, to obtain the expression vector pET22b (+) -G11-scFv-Nluc.
Example 2G11-scFv-Nluc fusion protein preparation
The preparation of the G11-scFv-Nluc fusion protein specifically comprises the following steps: based on the expression vector pET22b (+) -G11-scFv-Nluc in the step (1), transferring the whole expression vector 2uL into a competent cell of 100uLBL21(DE3), carrying out ice bath for 25min, carrying out water bath heat shock at 42 ℃ for 90s, and then rapidly putting the competent cell into the ice bath for 2 min; adding 1000uLLB culture medium, shaking at 37 deg.C for 1 hr, centrifuging at 5000rpm and 4 deg.C for 1min, collecting 300uL of supernatant, resuspending the strain, sucking 100uL, uniformly spreading onto solid culture medium containing carboxymethyl antibiotic, and inverting at 37 deg.C overnight. The next day, randomly selecting monoclonal bacteria, respectively inoculating to 2mLLB (containing carbenicillin 50ug/mL), culturing overnight, sucking 1mL of bacterial liquid the next day, and sending to Biotechnology engineering (Shanghai) GmbH for sequencing with sequencing primer T7.
100uL of the accurately sequenced positive monoclonal bacteria were inoculated into 10mLLB medium (containing 50ug/mL carbenicillin) and cultured overnight at 37 ℃ and 220 rpm. The next day, 10mL of the resulting suspension was transferred to 1LLB medium (containing 50ug/mL carbenicillin), and the mixture was subjected to amplification culture at 37 ℃ and 220rpm with shaking for 5 to 6 hours. Expression was induced overnight at 200rpm with the addition of IPTG inducer (0.5mM final concentration) at 30 ℃. The following day, bacteria were recovered by centrifugation at 10000rpm for 15min at 4 ℃ using a B-per bacterial lysate kit at 1: 4(g/mL) adding bacteria extract to extract protein, and adopting reduced Ni of Shanghai Biyuntian biotechnology limited+Purifying the target protein containing the His label by an affinity chromatography column, concentrating the protein in an ultrafiltration mode at 6000rpm, 15min and 4 ℃, and finally determining the molecular weight and the purity of the protein by adopting a Kangji SDS-PAGE kit. In SDSPAGE electrophoresis, 10% separation gel was used, and after electrophoresis at 60V for 30min, electrophoresis was carried out at 120V for 1 hour, see FIG. 2, where the bands areThe reference numeral 1 denotes a marker, the band 2(CL) denotes a bacterial lysate, the band 3(FT) denotes a loading flow-through, the bands 4 and 5(W1 and W7) denote washing solutions at the 1 st and 7 th tubes, and the bands 6 to 10(W8, 9, 11, 13 and 14) denote eluents at the 8 th, 9, 11, 13 and 14 th tubes, respectively, and the molecular weight of the fusion protein obtained from an electrophorogram is about 49 kDa.
Example 3 determination of a Glycocholic acid Standard Curve
1. Coating GCA-OVA: GCA-OVA concentration was diluted with carbonate buffer (pH 9.5) to 2.0 μ g/mL, 100 μ L/well coated in white opaque 96-well plates, incubated overnight at 4 ℃, and washed 5 times with PBST (phosphate buffer + 0.05% Tween 20).
2. Sealing the milk powder: after adding 3% skim milk powder, 300. mu.L/well and incubating at 37 ℃ for 1 hour, PBST (phosphate buffer + 0.05% Tween20) was washed 4 times.
3. And (3) competitive reaction: 50 μ L/well of glycocholic acid standard to be tested (0.01, 0.05, 0.1, 1, 10, 50, 1001000ng/mL) was added, along with 50 μ L/well of 2ug/mLG11-scFv-Nluc fusion protein, incubated at 37 ℃ for 1 hour, and washed 4 times with PBST (phosphate buffer + 0.05% Tween 20).
4. And (3) determination: the substrate solution was prepared by adding 25uL of substrate buffer Nano-GloLuciferaceasaybuffer (containing 0.5uL of luciferase substrate Nano-Gloassaystub) to 75uLPBS, and then 100 uL/well was added to the wells of the plate reader, and the chemiluminescence at 450nm was immediately measured using a plate reader.
5. Drawing a bioluminescence immunoassay standard curve: using origin8.5 software, RLU as ordinate and glycocholic acid standard solution concentration as abscissa, a four-parameter curve was fitted to obtain a standard inhibition curve of glycocholic acid for simulated enzyme-linked immunoassay, see fig. 3.
Example 4
A bioluminescent immunoassay kit for detecting glycocholic acid comprises G11-scFv-Nluc bifunctional active fusion protein, GCA-OVA and glycocholic acid provided by embodiments 1 to 3, and specifically comprises: the fusion protein (G11-scFv-Nluc) is used as a detection antibody, the glycocholic acid is used as a standard substance to establish a bioluminescence enzyme-linked immunoassay method, the method is high in specificity and good in sensitivity, the minimum detection limit is 0.131ug/mL, the sensitivity is improved by 38 times compared with that of a common enzyme-linked immunoassay method (5.08ug/mL), and the method provides an accurate, reliable and high-sensitivity detection method for rapidly detecting glycocholic acid in urine.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Guangdong university of industry
<120> G11-scFv-Nluc bifunctional active fusion protein and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1173
<212> DNA
<213> SNF1
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gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gatcacctgc 60
tccgggggtg gcagctatgc tggaagttac tattatggct ggtaccagca gaagtctcct 120
ggcagtgccc ctgtcactgt gatctatcaa aacaccaaga gaccctcgaa catcccttca 180
cgattctccg gttccctatc cggctccaca aacacattaa ccatcactgg ggtccaagtc 240
gaggacgagg ctgtctattt ctgtgggagc tgggaaggca gcactaatac tggctatgtt 300
ggtatatttg gggccgggac aaccctgacc gtcctaggtc agtcctctag atcttccgcc 360
gtgacgttgg acgagtccgg gggcggcctc cagacgcccg gaggaacgct cagcctcgtc 420
gccctgactc agccgtcctc ggtgtcagca aacccaggag aaaccgtcaa gatcacctgc 480
tccgggggtg gcagctatgc tggaagttac tattatggct ggtaccagca gaagtctcct 540
ggcagtgccc ctgtcactgt gatctatcaa aacaccaaga gaccctcgaa catcccttca 600
cgattctccg gttccctatc cggctccaca aacacattaa ccatcactgg ggtccaagtc 660
gaggacgagg ctgtctattt ctgtgggagc tgggaaggca gcactaatac tggctatgtt 720
ggtatatttg gggccgggac aaccctgacc gtcctaggtc agtcctctag atcttccgcc 780
gtgacgttgg acgagtccgg gggcggcctc cagacgcccg gaggaacgct cagcctcgtc 840
tgcaagggct ccgggttcac cttcagcagt tatggcatgg agtgggtgcg acaggcgccc 900
ggcaaggggc tggaatgggt cgctggtatc aacactggca gtagatacat atcttacgcg 960
acagcggtga agggccgtgc caccatctcg agggacgacg ggcagagcac tctgaggctg 1020
cagctgaaca acctcagggc tgaggacacc ggcacctact tctgcgccaa gaccggtggt 1080
ggtcgctgtg gcggtgctga tgctgcttgt atcgccgcat ggggccacgg gaccgaagtc 1140
atcgtctcct ccactagtgg ccaggccggc cag 1173
<210> 2
<211> 512
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atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60
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actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180
atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240
gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300
atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360
gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420
gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480
accggctggc ggctgtgcga acgcattctg gc 512
Claims (6)
1. A G11-scFv-Nluc fusion protein with bifunctional activity, which is characterized in that the G11-scFv-Nluc fusion protein is prepared by the following method:
the nano-luciferase is used as a bioluminescent catalyst, and an anti-glycocholic acid single-chain antibody (G11-scFv) is fused with the nano-luciferase and cloned into a pET22b expression vector to obtain the antibody.
2. The G11-scFv-Nluc bifunctional fusion protein of claim 1, wherein the G11-scFv-Nluc fusion protein is prepared by:
(1) construction of G11-scFv-Nluc expression vector: cloning G11-scFv and Nluc genes by a PCR technology, splicing to obtain a fusion gene G11-scFv-Nluc, and obtaining an expression vector pET22b (+) -G11-scFv-Nluc by double enzyme digestion, gene recovery and enzyme connection;
(2) preparation of G11-scFv-Nluc fusion protein: the expression vector is transformed into a competent cell BL21 by a chemical transformation method, then the positive monoclonal bacteria are cultured, and the fusion protein is induced and expressed by an inducer.
3. The G11-scFv-Nluc bifunctional fusion protein as claimed in claim 1, wherein the G11-scFv-Nluc expression vector of step (1) is constructed by the following steps:
cloning G11-scFv and Nluc genes respectively by taking SNF1, SNR1, SNF2 and SNR2 as primers;
performing overlap extension PCR technology by respectively taking the first round products G11-scFv and Nluc as templates and taking SNF1 and SNR2 as primers to obtain G11-scFv-Nluc;
and carrying out XholI and EcoRI double enzyme digestion on the pET22b vector and the G11scFv-Nluc gene respectively by using a second round of PCR, recovering the enzyme digestion product by cutting glue, and carrying out enzyme ligation again to obtain an expression vector pET22b (+) -G11-scFv-Nluc.
4. The G11-scFv-Nluc bifunctional fusion protein as claimed in claim 1, wherein the G11-scFv-Nluc fusion protein of step (2) is prepared by the following steps:
based on the expression vector pET22b (+) -G11-scFv-Nluc in the step (1), the whole expression vector is transferred into 100uLBL2 competent cells, ice bath is carried out for 25min, water bath heat shock is carried out for 90s at 42 ℃, and then the cells are quickly placed into an ice box for 2 min; adding 700uLLB culture medium, shaking at 37 ℃ for 1 hour, sucking a proper volume, and uniformly spreading the volume to a solid culture medium containing antibiotics for overnight culture; the next day, monoclonal bacteria are selected for culture, samples are sent for sequencing, a sequencing primer is T7, the monoclonal bacteria with positive sequencing accuracy are subjected to amplification culture, IPTG inducer is added for induction expression, B-per bacterial lysate is used for extracting protein, target protein is purified through a nickel affinity chromatography column, the protein is concentrated through ultrafiltration, and finally SDS-PAGE electrophoresis is adopted for determining the molecular weight and the purity of the protein.
5. Use of the G11-scFv-Nluc bifunctional active fusion protein of claim 1 as an antibody for detection of glycocholic acid in urine.
6. A bioluminescent immunoassay kit for detecting glycocholic acid, comprising the G11-scFv-Nluc bifunctional fusion protein of claim 1, GCA-OVA and glycocholic acid.
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| CN105018552A (en) * | 2015-07-20 | 2015-11-04 | 中国科学院海洋研究所 | Preparation method of fusion fluorescent protein in escherichia coli |
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