CN110240644B

CN110240644B - Human growth hormone receptor mutant, human growth hormone immunogen, polyclonal antibody and detection kit

Info

Publication number: CN110240644B
Application number: CN201910578251.0A
Authority: CN
Inventors: 林晓涛; 钱纯亘; 胡鹍辉
Original assignee: Shenzhen Yhlo Biotech Co Ltd
Current assignee: Shenzhen Yhlo Biotech Co Ltd
Priority date: 2019-06-28
Filing date: 2019-06-28
Publication date: 2021-03-16
Anticipated expiration: 2039-06-28
Also published as: CN110240644A

Abstract

The invention relates to a human growth hormone receptor mutant, a human growth hormone immunogen, a polyclonal antibody and a detection kit. The amino acid sequence of the extracellular segment of the human growth hormone receptor mutant is shown as SEQ ID No. 1. The human growth hormone receptor mutant has strong specific binding force with human growth hormone, and when the growth hormone receptor mutant is applied to preparing products for detecting the growth hormone, the specificity is good, and the sensitivity is high.

Description

Human growth hormone receptor mutant, human growth hormone immunogen, polyclonal antibody and detection kit

Technical Field

The invention relates to the field of biological detection, in particular to a human growth hormone receptor mutant, a human growth hormone immunogen, a polyclonal antibody and a detection kit.

Background

Human Growth Hormone (human Growth Hormone) is a single peptide chain protein secreted by anterior pituitary eosinophils, and mature human Growth Hormone consists of 191 amino acids with 4 cysteines in the chain, forming two pairs of disulfide bonds (C54-C165, C182-C189).

The growth hormone acts on the whole body, has biological activity of directly promoting growth, has effects on protein synthesis, glycometabolism and the like, has wide physiological effects, can act on bones and cartilages to cause the growth of height, and can promote the protein synthesis and lipolysis of a human body and regulate the water-salt balance in the body.

The clinical abnormality of growth hormone is mainly manifested in two aspects of growth hormone excess or growth hormone deficiency. Excessive growth hormone mainly causes excessive growth of bones, soft tissues, internal organs and the like, is manifested as Gigantism (Gigantism) in adolescent and acromegaly in adulthood, has various clinical manifestations, can cause acrohyperplasia such as skull thickening and the like, has rough face, has high cheekbones and other facial features, and can also cause obvious metabolic, cardiovascular and respiratory diseases. Deficient secretion of growth hormone can cause stunting of the body, resulting in dwarfism and dwarfism.

At present, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLI), and the like are commonly used as detection methods for growth hormone. These methods of growth hormone detection rely primarily on the ability of growth hormone antibodies to specifically bind to growth hormone in the test sample. Therefore, the specificity and sensitivity of growth hormone antibodies are important for the detection of growth hormone. However, the specificity and sensitivity of the current products for detecting growth hormone in the market are low, and the market demands cannot be met.

Disclosure of Invention

Based on this, there is a need for a human growth hormone receptor mutant which can specifically recognize human growth hormone and has good specificity and strong affinity. When the growth hormone receptor mutant is applied to the preparation of a detection reagent or a detection kit of human growth hormone, the specificity is good, and the sensitivity is high.

The human growth hormone receptor mutant is characterized in that the amino acid sequence of the extracellular segment of the human growth hormone receptor mutant is shown as SEQ ID No. 1.

The human growth hormone receptor mutant can specifically recognize human growth hormone, and has good specificity and strong affinity, and when the human growth hormone receptor mutant is applied to the preparation of a detection reagent or a detection kit of the human growth hormone, the human growth hormone receptor mutant has good specificity and high sensitivity.

A preparation method of a human growth hormone receptor mutant comprises the following steps:

introducing a target expression fragment into an empty vector to obtain an expression vector, wherein the target expression fragment is used for expressing a polypeptide with an amino acid sequence shown as SEQ ID No. 1; and

and transfecting the expression vector into a host cell for expression to obtain the human growth hormone receptor mutant.

A human growth hormone immunogen, the human growth hormone immunogen comprises a protein fragment with an amino acid sequence shown as SEQ ID No. 2.

A polyclonal antibody to human growth hormone derived from an animal immunized with the human growth hormone immunogen of claim 3.

A human growth hormone detection kit comprises the human growth hormone receptor mutant or the human growth hormone receptor mutant prepared by the preparation method of the human growth hormone receptor mutant; and/or

At least one of a first polyclonal antibody, a second polyclonal antibody and a third polyclonal antibody, wherein the first polyclonal antibody is obtained by immunizing animals by taking a protein fragment with an amino acid sequence shown as SEQ ID No.2 as immunogen, the second polyclonal antibody is obtained by taking a protein fragment with an amino acid sequence shown as SEQ ID No.3 as immunogen, and the third polyclonal antibody is obtained by immunizing animals by taking a protein fragment with an amino acid sequence shown as SEQ ID No.7 as immunogen.

In one embodiment, the first polyclonal antibody and the second polyclonal antibody are included at the same time, and the mass ratio of the first polyclonal antibody to the second polyclonal antibody is 1:1 to 2.5; or

The polyclonal antibody comprises the second polyclonal antibody and the third polyclonal antibody, wherein the mass ratio of the third polyclonal antibody to the second polyclonal antibody is 1:1 to 2.5.

In one embodiment, the first polyclonal antibody and the second polyclonal antibody are labeled with chemiluminescent labels.

In one embodiment, the recombinant human growth hormone receptor mutant further comprises a solid phase carrier, wherein the solid phase carrier is combined with the human growth hormone receptor mutant.

In one embodiment, the solid phase carrier is selected from one of magnetic microspheres, latex microspheres, polystyrene plates and nitrocellulose membranes.

In one embodiment, the kit further comprises at least one of a human growth hormone standard, a sample diluent, a coating solution, a confining solution, a washing solution and a developing solution.

Drawings

FIG. 1 is a map of the vector pFastBac1 of example 1;

FIG. 2 is a SDS-PAGE result of the human full-length growth hormone and the human full-length growth hormone mutant of example 3;

FIG. 3 is a SDS-PAGE result chart of the human growth hormone C-terminal fragment, the human growth hormone C-terminal fragment mutant, the human growth hormone N-terminal fragment and the human growth hormone N-terminal fragment mutant of example 3;

FIG. 4 is a SDS-PAGE result of the human growth hormone receptor of example 3;

FIGS. 5 to 7 are SDS-PAGE results of the polyclonal antibody of example 5;

FIG. 8 is a SDS-PAGE result of the human growth hormone receptor mutant of example 6;

FIG. 9 is a standard curve of the human growth hormone test kit of example 9.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

An embodiment of the present invention provides a human growth hormone receptor mutant, which has strong binding force with human growth hormone specificity and can be applied to preparation of a detection reagent for detecting growth hormone and a detection kit for detecting growth hormone.

The human Growth hormone receptor (Growth hormone receptor) gene is located on chromosome five and consists of 638 amino acids, wherein the first 18 amino acids are signal peptides. The mature human GHR molecule is a single chain glycoprotein with 620 amino acids. Wherein 246 amino acids at the N end of the human growth hormone receptor contain 5 potential glycosylation sites and are positioned outside cells to form a binding structure domain of the human growth hormone, 247 th to 270 th amino acids are transmembrane regions formed by strong hydrophobic amino acids, and 350 amino acids at the C end of the human growth hormone receptor are positioned inside cells to form a signal transduction structure domain. The human hormone receptor extracellular domain can specifically bind to human growth hormone.

The human growth hormone receptor mutant of the present embodiment includes a fragment in which mutations of N40G, N236G, and C259A have been introduced into the extracellular domain (binding domain) of human growth hormone receptor.

Specifically, the amino acid sequence of the extracellular segment of the human growth hormone receptor mutant is shown as SEQ ID No. 1. The amino acid sequence shown as SEQ ID NO.1 is: MFSGSEATAAILSRAPWSLQSVGPGLKTNSSKEPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKC FSVDEIVQPDPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSVPVYSLKVDKEYEVRVRSKQRGSGNYGEFSEVLYVTLPQMSQFTAEEDFY are provided.

In one embodiment, the extracellular segment of the human growth hormone receptor mutant is further linked to a receptor fusion tag at the C-terminus. The setting of the receptor fusion label is convenient for purifying the human growth hormone receptor mutant in the process of preparing the human growth hormone receptor mutant. Further, the acceptor fusion tag is 3 × flag. It is understood that the receptor fusion tag is not limited to 3 × flag, and in some embodiments, may be other fusion tags that facilitate purification of human growth hormone receptor mutants.

In one embodiment, the mutant human growth hormone receptor further comprises at least one of a transmembrane region of a wild-type human growth hormone receptor and an intracellular fragment of a wild-type human growth hormone receptor.

In one embodiment, the human growth hormone receptor mutant is a protein fragment with an amino acid sequence shown as SEQ ID No. 1.

In one embodiment, the human growth hormone receptor mutant is a protein fragment with a receptor fusion tag connected to the C terminal, and the amino acid sequence of the protein fragment is shown as SEQ ID No. 1. Further, the acceptor fusion tag is 3 × flag. Of course, the receptor fusion tag is not limited to 3 × flag, and in some embodiments, may be other fusion tags.

One embodiment of the invention provides a human growth hormone immunogen for preparing a polyclonal antibody of human growth hormone.

Specifically, the human growth hormone immunogen comprises a protein fragment with an amino acid sequence shown as SEQ ID No. 2. The protein fragment with the amino acid sequence shown as SEQ ID No.2 is obtained by C190A and C215A mutation at the C end of human growth hormone. The amino acid sequence shown as SEQ ID NO.2 is: YDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYAFRKDMDKVETFLRIVQCRSVEGSAGF are provided.

In one embodiment, the human growth hormone immunogen is a protein fragment with an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the human growth hormone immunogen is a protein fragment with a hormone fusion tag connected to the N end, and the amino acid sequence of the protein fragment is shown as SEQ ID No. 2. The hormone fusion label is arranged, so that the human growth hormone immunogen can be purified conveniently in the process of preparing the human growth hormone immunogen. Further, the hormone fusion tag is a His tag. Of course, the hormone fusion tag is not limited to 3 × flag, but in some embodiments, it may be any other fusion tag that facilitates purification of human growth hormone immunogen.

The research of the application shows that the traditional human growth hormone immunogen can not recognize some sites due to the structural problem, so that the diversity of the obtained polyclonal antibody is not rich enough. The human growth hormone immunogen is obtained by mutating (C190A and C215A) the polypeptide fragments from 136 th to 217 th positions of the C end of human growth hormone, and a polyclonal antibody obtained by taking the polypeptide fragments as immunogen can specifically recognize more sites of human growth hormone and has strong affinity.

The human growth hormone receptor mutant and the growth hormone immunogen can be prepared by a preparation method of recombinant protein. Specifically, a target expression fragment is introduced into an empty vector to obtain an expression vector; and then introducing the expression vector into an expression system for expression, thereby obtaining the protein expressed by the target expression fragment.

An embodiment of the present invention further provides a method for preparing a recombinant protein, which includes steps S110 to S150.

And step S110, introducing the target expression fragment into an empty vector to obtain an expression vector.

Specifically, the expression fragment of interest includes at least one of a nucleotide sequence of a human growth hormone receptor, a nucleotide sequence of a human growth hormone receptor mutant, and a nucleotide sequence of a human growth hormone immunogen. The target expression fragment is used for expressing at least one of human growth hormone immunogen, human growth hormone receptor and human growth hormone receptor mutant.

Specifically, the nucleotide sequence of the human growth hormone receptor mutant comprises a nucleotide sequence for expressing a protein fragment with an amino acid sequence shown as SEQ ID No. 1; the nucleotide sequence of the human growth hormone immunogen comprises a nucleotide sequence of a protein fragment with an expression amino acid sequence shown as SEQ ID No. 2. The nucleotide sequence of the human growth hormone receptor comprises a nucleotide sequence for expressing a protein fragment with an amino acid sequence shown as SEQ ID No. 9.

In one embodiment, the nucleotide sequence of the human growth hormone receptor mutant further comprises a nucleotide sequence for expressing a receptor fusion tag. The nucleotide sequence of the expression receptor fusion tag is arranged on the nucleotide sequence of the human growth hormone receptor mutant, so that the purification of the human growth hormone receptor mutant is facilitated. The receptor fusion tag is positioned at the N-terminal of the human growth hormone receptor mutant. Further, the receptor fusion tag is a 3 × flag tag. The receptor fusion tag is a polypeptide fragment with an amino acid sequence shown as SEQ ID No.4, and is positioned at the N end of a protein fragment with an amino acid sequence shown as SEQ ID No. 1. The amino acid sequence shown as SEQ ID No.4 is as follows: DYKDHDGDYKDHDIDYKDDDDK are provided.

In one embodiment, the nucleotide sequence of the human growth hormone immunogen further comprises a nucleotide sequence expressing a hormone fusion tag. The nucleotide sequence of the human growth hormone immunogen is provided with the nucleotide sequence of the expression hormone fusion tag, so that the purification of the human growth hormone immunogen is facilitated. The hormone fusion tag is positioned at the C terminal of the human growth hormone immunogen. Further, the hormone fusion tag is a His tag. The His tag is a polypeptide fragment formed by 6-10 histidines. In one embodiment, the amino acid sequence of the hormone fusion tag is HHHHHHHHHH.

In one embodiment, the empty vector is pFastBac 1. Of course, the empty vector is not limited to pFastBac1, but may be other vectors that can be used for expression.

In one embodiment, the expression fragment of interest comprises a nucleotide sequence of a human growth hormone receptor, a nucleotide sequence of a human growth hormone receptor mutant, and a nucleotide sequence of a human growth hormone immunogen. Of course, it is understood that in other embodiments, the human growth hormone receptor mutant, human growth hormone receptor and human growth hormone immunogen may be expressed separately in separate vectors.

S130, transfecting the expression vector into a host cell for expression to obtain a crude product.

Specifically, the expression vector is transfected into insect cells for expression to obtain a crude product. The specific operation includes S131 to S137.

S131, extracting the bacmid.

Specifically, the expression vector is transformed into a competent cell, coated into a blue-white screening plate, cultured overnight at 37 ℃, a white monoclonal is picked and inoculated into an LB medium, and then cultured overnight at 37 ℃ and 180-220 rpm, and then the thalli are collected and bacmid is extracted.

Further, the competent cell was DH10 Bac. Of course, the competent cells are not limited to DH10Bac, but may be other competent cells commonly used in the art.

S133, transfection.

Specifically, the density of Sf9 cells in the cell culture plate was adjusted and the viability of Sf9 cells was confirmed so that the density of Sf9 cells was 1.5X 10⁶2.5X 10 to one/mL⁶Cell viability greater than 95% (confirmed by trypan blue staining). Then uniformly mixing bacmid and transfection working solution, then incubating Sf9 cells instead of a culture medium, removing the transfection solution after 5-8 h, adding the cell culture medium into 2.0-3.0 mL/hole for culture, regularly confirming virus infection signs under a microscope, collecting culture supernatant after 64-76 h, centrifuging, and then discarding precipitates to obtain the P1 baculovirus.

And S135, virus amplification.

The density of Sf9 cells was adjusted and the viability of Sf9 cells was confirmed so that the density of Sf9 cells was 1X 10⁶2.5X 10 to one/mL⁶Cell viability greater than 95% (confirmed by trypan blue staining). Then adding P1 baculovirus solution into 30-50 mul/well for culturing, confirming virus infection sign under a regular microscope, collecting culture supernatant after 64-76 h, centrifuging and discarding precipitate to obtain P2 baculovirus. P2 baculovirus was added to Sf9 cells and cultured in a similar manner to obtain P3 baculovirus.

S137, recombinant protein expression.

According to 2X 10⁶Inoculating 500-1000 mL of culture medium at the density of each mL, and then performing suspension culture on the High Five cells by using a constant-temperature shaking table at the rotating speed of 150-180 rpm; after suspension culture for 6-8 h, adding 10-20 mL of the P3 baculovirus, continuing to culture for 54-62 h, and then centrifuging to collect High Five cells. Then, the expression of the collected High Five cells was confirmed.

Further, the step of confirming the expression of the collected High Five cells comprises: and taking about 1mL of cell suspension after expression, centrifuging, discarding the precipitate, adding 1 × electrophoresis loading buffer, performing SDS-PAGE, detecting by Western blot by using His-tag and flag-tag labeled antibodies, and confirming the expression condition of the protein.

Of course, the host cell is not limited to insect cells, and in other embodiments, the host cell may be other cells for expression. For example, Escherichia coli, yeast, etc.

And step S150, purifying the crude product.

Specifically, the human growth hormone immunogen is fused with a histidine tag, and is purified by Ni-sepharose FF affinity chromatography, which comprises the following specific operations:

taking the collected High Five cells, adding lysis buffer solution with the volume 5-10 times of the wet weight of the cells, carrying out ultrasonic lysis after heavy suspension, centrifuging for 30-60 min at 12000-15000 rpm and 4-12 ℃ after lysis, and then keeping supernatant; the supernatant was then filtered through a 0.22uM filter and then subjected to Ni-sepharose FF metal centrifugation chelate affinity chromatography for purification by continuous gradient competitive elution. Then, samples after Ni-sepharose FF affinity chromatography purification are collected after being identified by SDS-PAGE and Western Blot, and are dialyzed by dialysis buffer solution, so as to obtain the purified human growth hormone immunogen. Wherein the lysis buffer comprises 10 mM-50 mM Tris-Cl, 150 mM-500 mM NaCl, 0.5 mM-1.0 mM PMSF and 5% -10% (v/v) Glycerol, and the pH value is 7.2-8.0. The ultrasonic conditions are as follows: working for 4-8 seconds at 4-12 ℃, pausing for 10-15 seconds, and working for 10-15 minutes. The binding buffer solution for Ni-sepharose FF metal centrifugal chelating affinity chromatography comprises 10 mM-50 mM Tris-Cl, 150 mM-500 mM NaCl and 5% -10% (v/v) Glycerol, and the pH value is 7.2-8.0; the elution buffer solution for Ni-sepharose FF metal centrifugal chelating affinity chromatography comprises 10 mM-50 mM Tris-Cl, 150 mM-500 mM NaCl, 400 mM-550 mM Imidazole and 5% -10% (v/v) Glycerol, and the pH is 7.2-8.0. The dialysis buffer comprises PBS, and the pH value is 7.2-7.5.

The human growth hormone receptor mutant is fused with a 3 × flag tag, and is purified by M2bead affinity chromatography, and the specific operation is as follows:

taking the collected High Five cells, adding lysis buffer solution with the volume 5-10 times of the wet weight of the cells, carrying out ultrasonic lysis after heavy suspension, centrifuging for 30-60 min at 12000-15000 rpm and 4-12 ℃ after lysis, and then keeping supernatant; the supernatant was then filtered through a 0.22uM filter and subjected to M2bead affinity chromatography, followed by loading and washing, and then eluted with low pH glycine. The eluted sample is neutralized to pH 7.5 by 1M Tris-Cl pH9.0, and then the sample after M2bead affinity chromatography purification is collected after SDS-PAGE and Western Blot identification and is dialyzed by dialysis buffer solution, thus obtaining the purified human growth hormone receptor mutant. Wherein the lysis buffer comprises 10 mM-50 mM Tris-Cl, 150 mM-500 mM NaCl, 0.5 mM-1 mM PMSF and 5% -10% (v/v) Glycerol, and the pH value is 7.2-8.0. The ultrasonic conditions are as follows: working for 4-8 seconds at 4-12 ℃, pausing for 10-15 seconds, and working for 10-15 minutes. The binding buffer solution for M2bead affinity chromatography comprises 10 mM-50 mM Tris-Cl, 150 mM-500 mM NaCl and 5% -10% (v/v) Glycerol, and the pH value is 7.2-8.0; the elution buffer solution for M2bead affinity chromatography comprises 100mM Glycine and 5-10% (v/v) Glycerol, and the pH value is 3.0-3.5. The dialysis buffer comprises PBS, and the pH value is 7.2-7.5.

The preparation method of the recombinant protein has high efficiency by arranging the human growth hormone receptor mutant and the human growth hormone immunogen protein fragment on the same expression vector for expression.

The invention also provides a polyclonal antibody of human growth hormone, which is obtained by immunizing animals with the human growth hormone immunogen.

Specifically, the method for producing a polyclonal antibody against human growth hormone comprises step S210 to step 230.

And step S210, immunizing the animal with the immunogen to obtain an immunized animal.

Specifically, the immunogen comprises a protein fragment with an amino acid sequence shown as SEQ ID NO.2, a protein fragment with an amino acid sequence shown as SEQ ID NO.3, a protein fragment with an amino acid sequence shown as SEQ ID NO.5, a protein fragment with an amino acid sequence shown as SEQ ID NO.6, a protein fragment with an amino acid sequence shown as SEQ ID NO.7 or a protein fragment with an amino acid sequence shown as SEQ ID NO. 8.

In one embodiment, rabbits are immunized multiple times with an immunogen to obtain immunized rabbits. The dosage of immunogen is 0.1 mg-1.0 mg in each immunization, and the interval time of immunization is 10 days-14 days. Preferably, the dose of immunogen per immunization is 0.3mg to 0.5 mg. Of course, sites of immunization include, but are not limited to, subcutaneous dorsal, subcutaneous abdominal, subcutaneous axillary, subcutaneous extremities.

Step S230, extracting and purifying the anti-leptin polyclonal antibody from the immunized animal.

Specifically, polyclonal antibodies against leptin are isolated and purified from the blood of the immunized animal.

The preparation method of the anti-leptin polyclonal antibody is simple and convenient, and the polyclonal antibody of the human growth hormone can be obtained.

The invention also provides a human growth hormone detection kit, which comprises the human growth hormone receptor mutant, can be specifically combined with human growth hormone, and has good specificity and high detection sensitivity.

In one embodiment, the human growth hormone receptor mutant is used as a coated antibody for detecting growth hormone in a sample.

In one embodiment, the kit for detecting human growth hormone further comprises at least one of a first polyclonal antibody, a second polyclonal antibody and a third polyclonal antibody. The first polyclonal antibody is obtained by immunizing animals by taking a protein fragment with an amino acid sequence shown as SEQ ID No.2 as immunogen; the second polyclonal antibody is obtained by taking a protein fragment with an amino acid sequence shown as SEQ ID No.3 as an immunogen to immunize an animal; the third polyclonal antibody is obtained by immunizing animals by taking a protein fragment with an amino acid sequence shown as SEQ ID No.7 as immunogen.

The first polyclonal antibody, the second polyclonal antibody and the third polyclonal antibody are all capable of specifically binding with the growth hormone. At least one of the first polyclonal antibody, the second polyclonal antibody and the third polyclonal antibody can be used as a detection antibody for detecting the growth hormone in the sample.

In one embodiment, when the first polyclonal antibody, the second polyclonal antibody and the third polyclonal antibody are used as the detection antibodies, the first polyclonal antibody, the second polyclonal antibody and the third polyclonal antibody are all labeled with markers. Further, the first polyclonal antibody, the second polyclonal antibody and the third polyclonal antibody are all labeled with chemiluminescent markers. Specifically, the chemiluminescent label is isoluminol, acridinium ester, acridinium sulfonamide, ruthenium terpyridyl, alkaline phosphatase or horseradish peroxidase. Of course, in other embodiments, other labels are possible, such as colloidal gold, biotin, and the like.

In one embodiment, the kit for detecting human growth hormone comprises a first polyclonal antibody and a second polyclonal antibody, wherein the mass ratio of the first polyclonal antibody to the second polyclonal antibody is 1:1 to 2.5. The matching of the first polyclonal antibody and the second polyclonal antibody enables the human growth hormone detection reagent to have higher sensitivity and good specificity. Further, the mass ratio of the first polyclonal antibody to the second polyclonal antibody is 1:1 to 1.5.

In one embodiment, the kit for detecting human growth hormone comprises a second polyclonal antibody and a third polyclonal antibody, wherein the mass ratio of the third polyclonal antibody to the second polyclonal antibody is 1:1 to 2.5. The third polyclonal antibody and the second polyclonal antibody are matched, so that the human growth hormone detection reagent is higher in sensitivity and good in specificity. Further, the mass ratio of the third polyclonal antibody to the second polyclonal antibody is 1:1 to 1.5.

In one embodiment, the above-mentioned human growth hormone detection kit further comprises a solid carrier, and the solid carrier can bind to the human growth hormone receptor mutant. Further, the solid phase carrier is magnetic microspheres, latex microspheres, polystyrene plates or nitrocellulose membranes.

In one embodiment, the kit for detecting human growth hormone further comprises at least one of a human growth hormone standard, a sample diluent, a coating solution, a confining solution, a washing solution and a developing solution.

Specifically, the sample diluent is at least one selected from the group consisting of a carbonate buffer, a PBS buffer, and a TBS buffer. Further, the sample diluent is one of a carbonate buffer and a PBS buffer. Of course, it will be appreciated that in other embodiments, the sample diluent may be other sample diluents as are common in the art.

Specifically, the coating solution is at least one selected from carbonate buffer, PBS buffer and TBS buffer. Further, the coating solution is selected from one of carbonate buffer solution and PBS. Of course, it is understood that in other embodiments, the coating solution may be other coating solutions commonly found in the art.

Specifically, the blocking solution is selected from one of 1% to 3% (v/v) BSA in PBS, TBS and MOPS.

Of course, it is understood that in other embodiments, the confining liquid may be other confining liquids commonly found in the art.

The washing solution is selected from at least one of PBST containing 0.05-0.2% (v/v) Tween-20, TBS containing 0.05-0.2% (v/v) Tween-20 and 10% Triton. Further, the washing solution is PBST (phosphate Tween buffer) containing 0.05% -0.1% (v/v) Tween-20. Of course, it is understood that in other embodiments, the washing solution may be other washing solutions commonly found in the art.

The developing solution is selected according to the type of the marker of the detection antibody.

The human growth hormone detection kit comprises the human growth hormone receptor mutant, and has high detection sensitivity and good specificity.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.

Example 1

Expression vector construction

(1) The amino acid sequences shown in Table 1 were ligated to fusion tags by Utility Biotechnology Inc., entrusted to Anhui, and then subjected to codon optimization and nucleotide sequence synthesis. Wherein, the N ends of the protein segments with the amino acid sequences shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 5-SEQ ID No.8 are respectively connected with His labels (the amino acid sequence is HHHHHHHHHH), and the C end of the protein segment with the amino acid sequence shown as SEQ ID No.9 is connected with 3 Xflag labels (the amino acid sequence is shown as SEQ ID No. 4: DYKDHDGDYKDHDIDYKDDDDK). The mutation sites in the amino acid sequences of Table 1 are shown in bold.

TABLE 1

(2) The 7 nucleotide sequences were obtained by restriction enzymes EcoRI and HindIII and then loaded into 7 vectors of pFastBac1 (the map of pFastBac1 is shown in FIG. 1) to obtain 7 expression clones.

(3) Carrying out the following operations on the 7 expression clones prepared in the step (2) respectively to obtain 7 expression plasmids: and (3) transforming the expression clone into an escherichia coli competent cell Top10, culturing at 37 ℃ overnight, selecting a single clone, inoculating the single clone into 5mL of LB culture medium, culturing at 37 ℃ and 180rpm overnight, collecting thalli and extracting plasmids to obtain the expression plasmid containing the target expression fragment.

Example 2

Insect cell system expression of recombinant proteins

The 7 expression plasmids prepared in example 1 were transfected into insect cells for expression, and the operation of each expression plasmid was as follows:

(1) extracting the bacmid: the expression plasmid is transformed into a competent cell DH10Bac, coated into a blue-white spot screening plate, cultured overnight at 37 ℃, white monoclone is picked and inoculated into 10mL LB culture medium, cultured at 37 ℃ and 180rpm overnight, and then thalli are collected and bacmid is extracted.

(2) Transfection: sf9 cells (Life Technology) were placed in a 6-well cell culture plate and cultured adherent to the wall at 28 ℃ to give Sf9 cells at a density of 2.0X 10⁶Per mL, trypan blue staining confirmed cell viability greater than 95%. Then uniformly mixing the bacmids obtained in the step (1) with a transfection working solution, then incubating Sf9 cells instead of a culture medium, removing the transfection solution after 6h, andthe cell culture medium was added at 2.5 mL/well, and then subjected to static culture in an incubator at 28 ℃ to confirm the sign of viral infection under a periodic microscope, and after 72 hours, the culture supernatant was collected, centrifuged, and the precipitate was discarded to obtain P1 baculovirus.

(3) And (3) virus amplification: sf9 cells (Life Technology) were placed in a 6-well cell culture plate and cultured adherent to the wall at 28 ℃ to give Sf9 cells at a density of 1X 10⁶Per mL, trypan blue staining confirmed cell viability greater than 95%. Then, the P1 baculovirus solution obtained in step (2) was added at 45. mu.L/well, and then, the mixture was subjected to static culture in an incubator at 28 ℃ and periodically subjected to microscopic confirmation of signs of viral infection, and after 72 hours, the culture supernatant was collected, centrifuged, and the precipitate was discarded to obtain P2 baculovirus. P2 baculovirus was added to Sf9 cells and cultured in a similar manner to obtain P3 baculovirus.

(4) Recombinant protein expression: according to 2X 10⁶Inoculating 800mL of High Five cells per mL, and then performing suspension culture in a constant-temperature shaking incubator at 28 ℃ at the rotating speed of 150 rpm; after 8h of suspension culture, 15mL of the P3 baculovirus obtained in the step (3) is added, the culture is continued for 56h, and High Five is collected by centrifugation to obtain the expressed High Five.

(5) And (3) taking about 1mL of the cell suspension expressed in the step (4), centrifuging, discarding the precipitate, adding 1 × electrophoresis loading buffer, performing SDS-PAGE, detecting by Western blot by using His-tag and flag-tag labeled antibodies, and confirming the expression condition of the target protein.

Example 3

Recombinant protein purification

(1) The human growth hormone C-terminal fragment (wild type), the human growth hormone C-terminal fragment mutant, the human full-length growth hormone (wild type), the human full-length growth hormone mutant, the human growth hormone N-terminal fragment (wild type) and the human growth hormone N-terminal fragment mutant are fused with histidine tags, so that the purification is carried out by Ni-sepharose FF affinity chromatography, and the specific operation is as follows:

taking the High Five cells collected in example 2, adding lysis buffer solution with the volume 10 times of the wet weight of the cells, and carrying out ultrasonic lysis after resuspension (4 ℃, working for 4 seconds, pausing for 10 seconds, and working for 10 minutes); after centrifugation (15000rpm, 4 ℃, 30min), the supernatant was retained, and after filtration through a 0.22. mu.M filter, Ni-sepharose FF metal chelate affinity chromatography was performed to purify the target protein by continuous gradient competitive elution. Wherein: the lysis buffer solution consists of Tris-Cl, NaCl, PMSF, Glycerol and water, wherein the concentration of each component is as follows: 20mM Tris-Cl, 500mM NaCl, 0.5mM PMSF, 5% (v/v) Glycerol, pH 7.5 of the lysis buffer. The binding buffer solution for Ni-sepharose FF metal centrifugation chelating affinity chromatography consists of Tris-Cl, NaCl, Glycerol and water, wherein the concentration of each component is as follows: 20mM Tris-Cl, 500mM NaCl, 5% (v/v) Glycerol, pH 7.5 in binding buffer. The elution buffer solution for Ni-sepharose FF metal centrifugation chelating affinity chromatography consists of Tris-Cl, NaCl, Imidazole, Glycerol and water, wherein the concentration of each component is as follows; 20mM Tris-Cl, 500mM NaCl, 500mM Imidazole, 5% (v/v) Glycerol, pH of elution buffer 7.5. The purified protein was identified by SDS-PAGE and Western Blot, collected, dialyzed against 1 XPBS buffer at pH7.4, and the dialyzed samples were stored at-80 ℃.

The SDS-PAGE results of the purified human growth hormone C-terminal fragment (wild type), human growth hormone C-terminal fragment mutant, human full-length growth hormone (wild type), human full-length growth hormone mutant, human growth hormone N-terminal fragment (wild type) and human growth hormone N-terminal fragment mutant are shown in FIGS. 2 and 3. In FIG. 2, WT is a human full-length growth hormone wild type, and MT is a human full-length growth hormone mutant type. In fig. 3. Lane M is marker, lane 1 is human growth hormone N-terminal fragment wild type, lane 2 is human growth hormone N-terminal fragment mutant, lane 3 is human growth hormone C-terminal fragment wild type, and lane 4 is human growth hormone C-terminal fragment mutant.

(2) The human growth hormone receptor fused with the 3 × flag tag was therefore purified by M2bead affinity chromatography, as follows:

the High Five cells collected in example 2 were added with 10 times of the wet cell volume of lysis buffer, after resuspension, sonication (4 ℃, 4 seconds on, 10 seconds off, 10 minutes on), centrifugation (15000rpm, 4 ℃, 30min) the supernatant was retained, and after filtration using 0.22 μ M filter, M2bead affinity chromatography was performed, and after loading and washing, elution was performed directly using low pH glycine. Wherein: the lysis buffer solution consists of Tris-Cl, NaCl, PMSF, Glycerol and water, wherein the concentration of each component is as follows: 20mM Tris-Cl, 500mM NaCl, 0.5mM PMSF, 5% (v/v) Glycerol, pH 7.5 of the lysis buffer. The binding buffer solution for M2bead affinity chromatography is composed of Tris-Cl, NaCl, Glycerol and water, wherein the concentration of each component is as follows: 20mM Tris-Cl, 500mM NaCl, 5% (v/v) Glycerol, pH 7.5 in binding buffer. The elution buffer for M2bead affinity chromatography consists of Glycine, Glycerol and water, wherein the concentration of each component is as follows: 100mM Glycine, 5% (v/v) Glycerol, pH of elution buffer 3.5.

The eluted sample was neutralized to pH 7.5 with 1M Tris-Cl pH9.0, the purified protein was identified by SDS-PAGE and Western Blot and collected, and then dialyzed against 1 XPBS buffer at pH7.4, and the dialyzed sample was stored at-80 ℃.

The SDS-PAGE results after purification of human growth hormone receptor are shown in FIG. 4. In FIG. 4, lane M is marker, and lane 1 is wild-type human growth hormone receptor.

Example 4

Preparation of polyclonal antibodies

Animal immunization: two New Zealand male rabbits, weighing 2.5 kg. Freund's complete adjuvant or Freund's incomplete adjuvant was purchased from Sigma. Collecting blood from the vein of the immune anterior ear, standing at 4 deg.C, collecting serum, packaging, and storing at-80 deg.C as negative control serum. In each immunization, 500 mu g of the human growth hormone C-terminal fragment, the human growth hormone C-terminal fragment mutant, the human full-length growth hormone mutant, the human growth hormone N-terminal fragment and the human growth hormone N-terminal fragment mutant which are obtained by purifying in the embodiment 3 are respectively diluted to 1.25mL by PBS buffer solution and mixed with 1.25mL of adjuvant, and emulsified by vortex oscillation for 100min to prepare 6 different immunogen injection solutions. The immunization procedure is shown in table 2.

TABLE 2

Wherein the ELISA detection of polyclonal antibody production by each immunogen is as follows:

(1) coating the immunogen: diluting the immunogen to 2 mu g/mL by using CB buffer solution (50mM carbonate buffer solution, pH 9.6: 0.75g sodium carbonate, 1.46g sodium bicarbonate and deionized water to a constant volume of 500mL), adding the diluted immunogen into an ELISA plate according to 100 uL/hole, and incubating overnight at 4 ℃;

(2) and (3) sealing: the coating solution was removed and 200uL of blocking solution (5% BSA, 1 XPBS, pH7.4) was added and incubated at 37 ℃ for 4 h.

(3) After removing the confining liquid, respectively adding 50 mu L of serum to be detected and negative control serum, simultaneously adding 50 mu L of goat anti-rabbit secondary antibody labeled by horseradish peroxidase into the diluted commercial product, and incubating for 1h at 37 ℃.

(4) After removing the reaction solution, using a cleaning solution (0.1% Tween-20, 1 XPBS, pH7.4) 200. mu.L cleaning, after 5 times of cleaning, adding 100. mu.L of commercial color solution TMB, incubating at 37 ℃ for 10min, adding 100. mu.L of 1N sulfuric acid solution to terminate the reaction;

(5) reading on a microplate reader.

Example 5

Purification of polyclonal antibodies

(1) According to the ELISA results of the sera after the 4 th immunization or the 5 th immunization of example 4, when the titer reached 1X 10⁶In time, rabbits were bled in bulk and centrifuged to collect serum.

(2) The serum volume was measured accurately, solid ammonium sulfate was added until the saturation reached 50%, and after stirring for 2h at 4 ℃, the supernatant was discarded after centrifugation (12000rpm, 30min, 4 ℃).

(3) The precipitate was reconstituted by adding 20mL of 1 XPBS, pH7.4, at a wet weight of 1g, and filtered through a 0.22. mu.M filter.

(4) rProteinA-sepharose FF affinity chromatography: 1) and (3) chromatographic column balancing: the column was equilibrated with 5 bed volumes of binding buffer (1 × PBS, pH 7.4). 2) Loading: the supernatant of the reconstituted and filtered polyclonal antibody was added at a flow rate of 1 mL/min. 3) Wash: after the loading was complete, the column was washed with 10 bed volumes of binding buffer (1 × PBS, pH 7.4). 4) And (3) elution: elution was performed by adding elution buffer (0.1M Glycine, pH 2.7) and after elution the samples were neutralized to pH 7.2 by adding 1M Tris-Cl pH9.0 buffer. 5) The eluted samples were collected after SDS-PAGE analysis and after Bradford assay the individual polyclonal antibodies were numbered, as shown in Table 3 below.

TABLE 3

Immunogens	Name number of polyclonal antibody corresponding to immunogen
		Human full-length growth hormone	pAb-01
Human full-length growth hormone mutant	pAb-02
		Human growth hormone N-terminal fragment	pAb-03
Human growth hormone N-terminal fragment mutant	pAb-04
		Human growth hormone C-terminal fragment	pAb-05
Human growth hormone C-terminal fragment mutant	pAb-06

The results of the purification of the polyclonal antibody (SDS-PAGE) are shown in FIGS. 5 to 7. In FIG. 6, lane M is marker, and lane 1 corresponds to polyclonal antibody (pAb-01) with human full-length growth hormone as an immunogen; lane 2 corresponds to polyclonal antibodies (pAb-02) produced by human full-length growth hormone mutants as immunogens. In FIG. 7, lane M is marker, and lane 3 corresponds to polyclonal antibody (pAb-03) having an N-terminal fragment of human growth hormone as an immunogen; lane 4 corresponds to polyclonal antibody (pAb-04) generated by the N-terminal fragment mutant of human growth hormone as an immunogen. In FIG. 8, lane M is marker, and lane 5 corresponds to polyclonal antibody (pAb-05) with a C-terminal fragment of human growth hormone as an immunogen; lane 6 corresponds to polyclonal antibody (pAb-06) generated by the C-terminal fragment mutant of human growth hormone as an immunogen.

Example 6

Screening of human growth hormone receptor mutants

(1) Various human growth hormone receptor mutants were prepared by constructing expression vectors, expressing in an insect cell expression system, and purifying according to the methods of examples 1 to 3. Among them, the mutation of some human growth hormone receptor mutants is shown in Table 4 below.

TABLE 4

Wherein, the amino acid sequence of the human growth hormone receptor mutant with N40G, N236G and C259A mutations is shown as SEQ ID No.1, and specifically comprises the following steps: MFSGSEATAAILSRAPWSLQSVGPGLKTNSSKEPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKCFSVDEIVQPDPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSVPVYSLKVDKEYEVRVRSKQRGSGNYGEFSEVLYVTLPQMSQFTAEEDFY are provided.

The electrophoretogram of the purified human growth hormone receptor mutant having mutations N40G, N236G and C259A is shown in fig. 8. In FIG. 8, lane M is marker, and lane 1 is a human growth hormone receptor mutant in which mutations of N40G, N236G and C259A have been introduced.

(2) After obtaining the human growth hormone receptor mutants, the human growth hormone receptor mutants are respectively diluted to 1 mu g/mL by using PBS buffer solution, then the human growth hormone receptor mutants are respectively used as coated capture antibodies, and positive serum samples with the growth hormone concentrations of 0.1ng/mL, 1ng/mL and 10ng/mL and 9 negative serum samples are detected. The specific operation of each human growth hormone receptor mutant is as follows:

coating human growth hormone receptor mutants: the human growth hormone receptor mutant is added into an enzyme label plate at 100 mu L/hole for 12 holes in total so as to facilitate the subsequent detection of samples. Then incubated at 37 ℃ for 30min and removed, followed by addition of 0.5% BSA in PBS per well, incubation at 4 ℃ for 16h, removal of the solution and drying.

Sample adding: 100 mul of positive serum samples and 9 negative serum samples with growth hormone concentration of 0.1ng/mL, 1ng/mL and 10ng/mL are respectively and correspondingly added into 12 enzyme-labeled wells coated with human growth hormone receptor mutants, incubated at 37 ℃ for 30min and then removed.

HRP-labeled polyclonal antibody: adding 100 μ L of HRP-labeled polyclonal antibody (HRP-labeled pAb-01) with concentration of 1mg/mL into each enzyme-labeled well after sample addition and incubation, incubating at 37 deg.C for 15min, and removing;

cleaning: PBST washing at 200. mu.L/time, washing for 6 times, adding 100. mu.L TMB, developing at 37 deg.C for 10min, adding 100. mu.L 1M H₂SO₄And a450 reading.

The results of testing some human growth hormone receptor mutants are shown in Table 5.

TABLE 5

As can be seen from Table 5, the human growth hormone receptor mutants (protein fragments having amino acid sequences shown in SEQ ID No. 1) mutated to N40G, N236G and C259A have the best binding effect with the wild type human growth hormone.

Example 7

Polyclonal antibody screening

The human growth hormone receptor mutants prepared in example 6 and mutated to N40G, N236G and C259A were used as coated capture antibodies, and polyclonal antibodies were screened using HRP-labeled polyclonal antibodies as detection antibodies. The specific operation is as follows:

coating human growth hormone receptor mutants: diluting human growth hormone receptor mutant to 1 mu g/mL by PBS buffer solution, then adding 100 mu L/well into an enzyme label plate, adding 90 wells in total, incubating at 37 ℃ for 30min, removing, adding PBS solution containing 0.5% BSA into each well, incubating at 4 ℃ for 16h, removing the solution, and drying;

sample adding: dividing the coated enzyme-labeled wells into 9 groups, 10 wells in each group, adding 100 microliter of positive serum samples with growth hormone concentrations of 0pg/mL, 0.5pg/mL, 1pg/mL, 5pg/mL, 10pg/mL, 15pg/mL, 20pg/mL, 30pg/mL and 50pg/mL into the coated enzyme-labeled wells to form 9 groups of test groups with different growth hormone concentrations, wherein the growth hormone concentrations in the same group are the same, and then removing the test groups after incubation at 37 ℃ for 30 min.

HRP-labeled polyclonal antibody: the following operations were performed for 9 groups of test groups with different growth hormone concentrations after removal of serum samples: mu.L of HRP-labeled pAb-01, pAb-02, pAb-03, pAb-04, pAb-05, pAb-06, pAb-01& pAb-04, pAb-01& pAb-06, pAb-04& pAb-06, and pAb-01& pAb-04& pAb-06, were added to each group, respectively, and were removed after incubation at 37 ℃ for 15 min. Wherein pAb-01& pAb-04 represents a mixed polyclonal antibody of pAb-01 and pAb-04, and the mixed polyclonal antibody is obtained by adjusting the concentrations of pAb-01 and pAb-04 to 1mg/mL by PBS buffer solution and then mixing the obtained solutions with equal mass; pAb-01& pAb-06 show a mixed polyclonal antibody of pAb-01 and pAb-06, and pAb-01 and pAb-06 were adjusted to a concentration of 1mg/mL in PBS buffer and mixed at equal mass. pAb-04& pAb-06, pAb-04& pAb-06 and pAb-01& pAb-04& pAb-06 are the same.

Cleaning: washing with PBS-T at a ratio of 200. mu.L/time for 6 times, adding 100. mu.L of TMB, developing at 37 deg.C for 10min, adding 100. mu.L of 1M H₂SO₄And a450 reading.

The results of the partial tests of the polyclonal antibody screening are shown in Table 6.

TABLE 6

As is clear from Table 6, when pAb-02 was used as the detection antibody, the signal value was greatly decreased, whereas when pAb-01& pAb-06 was used, the signal was stable and the linearity was most favorable.

Example 8

Preparation of human growth hormone detection kit

(1) Coating of human growth hormone receptor: taking 50mg of carboxylated magnetic particles (with the particle size of 0.5 mu m), carrying out magnetic separation, leaving a precipitate, then carrying out resuspension by using 20mM of MES buffer solution with the pH of 5.5, adding 1mL of freshly prepared 10mg/mL of EDC aqueous solution, activating the carboxyl groups on the surfaces of magnetic beads, adding 4mg of the human growth hormone receptor mutant prepared in example 6 (with the mutations of N40G, N236G and C259A and the amino acid sequence shown as SEQ ID No. 1), suspending at room temperature for 6h, carrying out magnetic separation, removing supernatant, carrying out resuspension to 1mg/mL by using 100mM Tris buffer with the pH of 8.0 and containing 2% BSA to obtain coated magnetic particles, subpackaging the coated magnetic particles with 5 mL/bottle, and storing the coated magnetic particles at the temperature of 4 ℃ for later use.

(2) Preparation of polyclonal antibody against acridinium ester-labeled human growth hormone: 50 μ L of 25mg/mL of the polyclonal antibody mixture purified in example 5 (pAb-01 and pAb-06 were mixed at a mass ratio of 1: 1), 150 μ L of 0.1M carbonate buffer pH9.0 was added, the mixture was mixed, 1.5 μ L of 5mg/mL acridine ester was added, the mixture was reacted at room temperature in the dark for 1.5h, the reaction mixture was desalted using a 5mL GE desaling prepacked column, a TBS equilibrium column was used, the reacted acridine ester solution was added, and a protein peak sample was collected and stored, the sample was dispensed at 5 mL/bottle, and the sample was stored at 4 ℃.

(3) Preparation of growth hormone standard: growth hormone standards (purchased from Abcam) were formulated with buffer (40mM Tris-Cl, 0.5% BSA, 1% NaCl, pH8.0) at concentrations of 0ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 20ng/mL, 25ng/mL, 30ng/mL, and were lyophilized at 1mL per vial and stored at 4 ℃ until needed.

Example 9

Performance evaluation of the human growth hormone detection kit:

the human growth hormone assay kit prepared in example 8 was used for performance evaluation by a double antibody sandwich method using a chemiluminescence analyzer as an assay tool. Reacting a sample to be detected or a human growth hormone standard substance with magnetic particles coated by a growth hormone receptor mutant for 10min, and carrying out magnetic separation. Then, a polyclonal antibody of acridinium ester-labeled human growth hormone was added, and after a reaction time of 10min, magnetic separation was performed. And then adding the chemiluminescence pre-excitation liquid and the chemiluminescence excitation liquid in sequence to carry out luminescence reaction, finally recording the luminescence intensity, drawing a standard curve, and calculating the content of the human growth hormone in the sample to be detected according to the standard curve. The standard curve of this example is shown in FIG. 9.

Detection of sensitivity:

the sensitivity of the human growth hormone detection kit of example 8 calculated with reference to the recommended experimental method of CLSI EP17-A document was 0.03 ng/mL.

And (3) linear detection:

the human growth hormone test kit of example 8 and human growth hormone standards with the concentration of 03ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 20ng/mL and 50ng/mL are used for linear analysis, and the linear correlation coefficient r is calculated to be 0.9991. The linear range of the human growth hormone detection kit in the example 8 calculated according to the experimental method recommended by CLSI EP17-A document is as follows: 0.03 ng/mL-50 ng/mL.

And (3) measuring precision:

two human growth hormone samples (prepared from human growth hormone standard substances purchased from Abcam) with the concentrations of 20ng/mL and 60ng/mL are taken, each concentration of each sample is respectively carried out in 3 parallel, three batches of the human growth hormone detection kit of the embodiment 8 are used for detection, and the intra-batch and inter-batch differences of the human growth hormone detection kit are calculated.

The calculation results show that the difference between batches and between batches of the human growth hormone detection kit in the embodiment 8 is less than 5 percent.

Interference experiments:

20 serum samples with the human growth hormone concentration of 5 ng/mL-30 ng/mL are taken, mixed according to the volume ratio of 1:1 to serve as mixed serum samples, the mixed serum samples are divided into six groups, five groups are respectively added with different interferents, and the rest group serves as a control. The added interferents are combined bilirubin, free bilirubin, hemoglobin, ascorbic acid, cholesterol and glyceride, and the mass ratio of the interferents to the mixed serum is 1: 20. then, the content of human growth hormone in the mixed serum and the content of human growth hormone in the mixed serum added with different interferents are respectively measured, and the deviation of the human growth hormone between the mixed serum and the mixed serum added with the interferents is calculated, wherein the deviation is within the acceptable range of +/-10%.

The calculation result shows that the interference of the human growth hormone detection kit in the embodiment 8 reaches the file standard of NCCLS, and the kit can be used for accurately evaluating the clinical human growth hormone condition.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Shenzhen Shenhuilong Biotech stock Co., Ltd

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Claims

1. The human growth hormone receptor mutant is characterized in that the amino acid sequence of the human growth hormone receptor mutant is shown as SEQ ID No. 1.

2. The method for preparing a human growth hormone receptor mutant according to claim 1, which comprises the steps of:

3. The method of claim 2, wherein the empty vector is pfastpac 1.

4. A kit for detecting human growth hormone, comprising the human growth hormone receptor mutant of claim 1.

5. The kit for detecting human growth hormone according to claim 4, further comprising a solid support, wherein the solid support is bound to the human growth hormone receptor mutant.

6. The kit for detecting human growth hormone of claim 5, wherein the solid phase carrier is selected from one of magnetic microspheres, latex microspheres, polystyrene plates and nitrocellulose membranes.

7. The human growth hormone detection kit according to any one of claims 4 to 6, further comprising at least one of a human growth hormone standard, a sample diluent, a coating solution, a confining solution, a washing solution and a developing solution.

8. The kit according to claim 4, wherein the kit further comprises a sample diluent, and the sample diluent is at least one selected from the group consisting of carbonate buffer, PBS buffer, and TBS buffer.

9. The kit according to claim 8, further comprising a coating solution selected from at least one of carbonate buffer, PBS buffer and TBS buffer.

10. The kit of claim 9, further comprising a washing solution selected from the group consisting of 0.05-0.2% (v/v) Tween-20 in PBST, 0.05-0.2% (v/v) Tween-20 in TBS, and 10% Triton.