CN112034175A

CN112034175A - ApoA-II and ApoC-II detection mixed quality control product and preparation method thereof

Info

Publication number: CN112034175A
Application number: CN202010262732.3A
Authority: CN
Inventors: 袁嘉扬; 吴炯; 戴谦
Original assignee: Suzhou Ecoris Biotechnology Co ltd
Current assignee: Suzhou Ecoris Biotechnology Co ltd
Priority date: 2020-04-03
Filing date: 2020-04-03
Publication date: 2020-12-04

Abstract

The invention provides an ApoA-II and ApoC-II detection mixed quality control product, which is a freeze-dried product of trihydroxymethylaminomethane, polyethylene glycol 6000, ProClin 950, deexcin serum and two protein positive substances, namely ApoA-II and ApoC-II. The preparation method of the ApoA-II and ApoC-II detection mixed quality control product comprises the following steps: (1) preparing human ApoA-II and ApoC-II gene clone and expression vector; (2) an 80L fermentation tank is utilized to ferment and express rhApoA-II and rhApoC-II in a large scale; (3) purifying rhApoA-II and rhApoC-II; (4) and (3) preparation, assignment and subpackage of ApoA-II and ApoC-II protein quality control products. The ApoA-II and ApoC-II detection mixed quality control product prepared by using the eukaryotic cell system has the advantages of good stability, low cost, simple and convenient use, contribution to animal experiments, no influence by transportation and temperature factors, good detection repeatability and high stability, can meet the quality control requirements of clinic on ApoA-II and ApoC-II detection, and can improve the accuracy of clinical sample detection results.

Description

ApoA-II and ApoC-II detection mixed quality control product and preparation method thereof

Technical Field

The invention relates to a detection quality control product, in particular to an ApoA-II and ApoC-II detection mixed quality control product and a preparation method thereof.

Background

Apolipoprotein is the protein part of plasma lipoprotein and can combine and transport blood fat to various tissues of the body for metabolism and utilization. A great deal of research finds that the mutation of the apolipoprotein gene forms different allelic polymorphism and further forms different phenotypes of the apolipoprotein, which can influence the metabolism and utilization of blood fat, thereby influencing the occurrence and development of hyperlipidemia, atherosclerosis, cardiovascular and cerebrovascular diseases and the like.

Apolipoproteins are mainly classified into A, B, C, D, E types, and their basic functions are to carry lipid substances and to stabilize lipoprotein structures, and some apolipoproteins also have the functions of activating lipoprotein-metabolizing enzymes, recognizing receptors, and the like. It is mainly synthesized in liver (part in small intestine), named according to ABC system, and each type can be subdivided into several subclasses, which are expressed by Roman numerals.

Apolipoproteins are important components constituting plasma lipoproteins, impart lipids in a soluble form, and play an important role in plasma lipoprotein metabolism: (1) promoting lipid transport; (2) modulating the activity of the enzyme; (3) leading plasma lipoproteins to bind to cell surface receptors is a functionally extremely active group of plasma proteins.

ApoA-II is a second most abundant apolipoprotein in HDL, 15% of which is HDL2 and 25% of which is HDL 3. CM represents 7% to 10% of total apolipoprotein, and VLDL is also present in small amount. The amino acid, cDNA and gene sequences of ApoA-II protein were elucidated until 1985. ApoA-II is composed of 77 amino acid residues of two polypeptide chains. ApoA-II was found to have a molecular weight of 17000D on SDS-PAGE without reducing agent and to exist as a dimer in human plasma. The molecular weight of the monomer of ApoA-II is 8700D. The C-terminal amino acid residue of the human ApoA-II protein is glutamic acid, the N-terminal amino acid residue is pyrrolidone acid, and histidine, arginine and tryptophan are lacked. ApoA-II is polymorphic, with the predominant polymorphism having an isoelectric point of 4.9, and the lesser polymorphic forms having isoelectric points of 5.17, 4.68, 4.42 and 4.20.

Apolipoprotein C is the major apolipoprotein of very low density lipoprotein cholesterol, and is also present in high density lipoprotein-cholesterol and low density lipoprotein-cholesterol, there are 3 different apolipoproteins C, ApoC-I, ApoC-II, ApoC-III, which are minor structural proteins of CM, VLDL, and HDL.

Currently, ApoA-II and ApoC-II detection mixed quality control substances have the following defects: poor stability, high cost, inconvenient use, unfavorable use for animal experiments, poor detection repeatability and poor accuracy of clinical sample detection results.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an ApoA-II and ApoC-II detection mixed quality control product and a preparation method thereof, which have the advantages of good stability, low cost, simple and convenient use, contribution to animal experiments, no influence of transportation and temperature factors, good detection repeatability, capability of meeting the quality control requirements of clinical detection on ApoA-II and ApoC-II, and capability of improving the accuracy of clinical sample detection results.

In order to solve the above technical problems, embodiments of the present invention provide an ApoA-II and ApoC-II detection mixed quality control product, which is a freeze-dried product including two protein positive substances, ApoA-II and ApoC-II, and ApoA-II serum.

Wherein, the ApoA-II and ApoC-II detection mixed quality control product also comprises trihydroxymethylaminomethane, polyethylene glycol 6000 and ProClin 950.

Preferably, the concentration of the tris is not less than 99.9%.

The invention also provides a preparation method of the ApoA-II and ApoC-II detection mixed quality control product, which comprises the following steps:

(1) preparing human ApoA-II and ApoC-II gene clone and expression vector;

(2) an 80L fermentation tank is utilized to ferment and express rhApoA-II and rhApoC-II in a large scale;

(3) purifying rhApoA-II and rhApoC-II;

(4) and (3) preparation, assignment and subpackage of ApoA-II and ApoC-II protein quality control products.

Wherein, the step (1) comprises the following steps:

(1-1) extracting total RNA from human liver tissue by Trizol method, cutting fresh separated human liver tissue into 100mg size, immediately putting into liquid nitrogen;

grinding frozen tissue 300mg, transferring the ground tissue into 50ml centrifuge tube, adding Trizol 5ml, homogenizing at high speed for 15-30sec at room temperature with homogenizer, adding chloroform 1.0ml, shaking, standing at room temperature for 5min, and centrifuging at 4 deg.C (12000rpm/min) for 15 min;

transferring the upper water phase into another centrifuge tube, adding isopropanol with the same volume, shaking up, precipitating for 10min, centrifuging at 4 deg.C (12000rpm/min) for 15min, discarding supernatant, adding 1ml of 75% ethanol, washing precipitate, centrifuging at 4 deg.C (12000rpm/min) for 5min, dissolving total RNA precipitate in 50ul DEPC treated water, and storing at-80 deg.C for use;

(1-2) taking 1ug of extracted RNA, 1ul of OligodT, denaturing at 70 ℃ for 10min, carrying out ice bath for 1min, and then adding the following reaction liquid:

the reaction was carried out in a PCR apparatus at 42 ℃ for 60min, at 95 ℃ for 5min, and cDNA was synthesized for the following reactions:

designing primers according to the published human ApoA-II and ApoC-II genes in Genebank,

poA-II-F：ATGAAGCTGCTCCAGCAACTGT；

ApoA-II-R：TTATCACTGGGTGGCAGGCTGT；

ApoC-II-F：TCCAGCAGCAAGATTCAGAGTGCC；

ApoC-II-R：TGGGATGTCACCCTTCAGGGTC；

the reaction system is as follows:

amplification was according to the following PCR amplification procedure:

①94℃2min；

② 30 cycles of 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 30 sec;

③72℃10min；

carrying out 1% agarose electrophoresis on the PCR product, cutting down a target fragment under an ultraviolet lamp, and recovering by using a DNA recovery kit;

(1-3) connecting the recovered ApoA-II and ApoC-II gene fragments with a T carrier overnight at 16 ℃, wherein the connection reaction system is as follows:

finally, recombinant clone plasmids pMD18-T-ApoA-II and pMD18-T-ApoC-II are obtained;

(1-4)XL₁preparation of Blue competent cells

(1-4-1) selecting a single colony to be inoculated in 5ml of LB culture medium, carrying out shaking culture at 37 ℃ overnight, inoculating lml bacterial liquid into a 1L culture bottle containing 100ml of LB culture medium, and carrying out shaking culture at 37 ℃ and 250r/min for 3 h;

(1-4-2) centrifuging at 4 ℃ for 10min (4000r/min) and collecting cells;

(1-4-3) pouring out the culture solution, and inverting the tube for 1min to drain the culture solution;

(1-4-4) with 60ml of precooled 0.1mol/L CaCl₂-MgCl₂Resuspending the cells;

(1-4-5) centrifuging at 4 ℃ (4000r/min) for 10min, and recovering cells;

(1-4-6) adding 140ul DMSO, gently rotating and uniformly mixing, and standing on ice for 15 min;

(1-4-7) adding 140ul DMSO, gently rotating and mixing uniformly, standing on ice for 15min, subpackaging 100ul each tube aseptically, and freezing at-80 ℃;

(1-5) transformation of recombinant plasmid

(1-5-1) thawing a tube of frozen competent cells on ice, adding the ligation product, and gently mixing;

(1-5-2) carrying out ice bath for 30 min;

(1-5-3) putting the reaction tube into a water bath at 42 ℃ and thermally exciting for 90 s;

(1-5-4) adding 800ul of preheated SOC culture medium into the tube, transferring the tube to a shaking table for shake culture (225r/min) for 1h, so that bacteria are revived and the antibiotic resistance marker gene coded by the plasmid is expressed;

(1-5-5) spreading 200ul of transformed competent cells on LB plate medium containing Amp (100ug/ml), standing at room temperature until the liquid is absorbed, and culturing at 37 deg.C for 12-16 h in an inverted manner;

(1-6) construction of rhApoA-I Pichia pastoris secretory expression vector

(1-6-1) designing primers according to an hApoA-IDNA sequence (accession number: NM000039) published by GenBank, amplifying an ApoA-II and ApoC-II coding sequence, wherein an Xhol restriction endonuclease site and a Kex2 protease recognition sequence coding gene are introduced into the 5 'end, and an Xbal restriction endonuclease site is introduced into the 3' end;

an upstream primer: 31bp

5’-AAACTCGAGAAGAGAGATGAACCCCCCCAGA-3’；

A downstream primer: 32bp

5’-GCGTCTAGATCACTGGGTGTTGAGCTTCTTAG-3’；

(1-6-2) PCR reaction

A PCR reaction system was set up in 0.2ml EP tubes in the following proportions:

performing cyclic amplification on a PCR instrument, wherein the amplification procedure comprises the following steps:

①94℃4min；

② 30 cycles of 94 ℃ for 30s, 61 ℃ for 30s and 72 ℃ for 1 min;

③72℃10min；

carrying out 1% agarose gel electrophoresis on the amplification product, observing the size of the fragment, and determining whether a correct target gene is obtained;

(1-7) construction of pPICZa-hApoA-II and pPICZa-hApoC-II secretory expression vectors

Xhol and Xbal enzyme cutting sites are respectively designed in PCR upstream and downstream primers for constructing an expression vector, and are used for gene recombination to the corresponding endonuclease sites of the vector pPICZa;

digesting the target gene hApoA-I and the vector pPICZa by Xhol and Xbal, recovering an enzyme digestion product, and connecting overnight at 16 ℃;

(1-8) preparation of Yeast competence

(1-8-1) taking Pichia pastoris X-33 strain, streak-culturing on YPD negative culture plate without antibiotic at 30℃,

The bacterial colony grows to2-3 mm, selecting a single colony, inoculating the single colony in 5ml YPD medium, and performing shake culture at 225r/min and 30 ℃ for 24 h;

(1-8-2) inoculating 200ul of the bacterial liquid into 200ml of YPD culture medium, and performing shake culture at 250r/min and 30 ℃ overnight;

(1-8-3) centrifuging at 4 ℃ (3000r/min) for 5min, and collecting cells;

(1-8-4) pouring out the culture solution, and inverting the tube for 1min to drain the culture solution;

(1-8-5) resuspending the cells with 200ul of precooled ultrapure water, centrifuging at 4 ℃, and discarding the supernatant;

(1-8-6) resuspending the cells with 20ml of pre-cooled 1mol/L D-sorbitol, centrifuging at 4 ℃ for 5min (3000r/min), and discarding the supernatant;

(1-8-7) resuspending the cell pellet with 300ul of pre-cooled 1mol/L D-sorbitol, gently rotating and mixing, placing on ice for use on the same day;

(1-9) transformation

(1-9-1) taking 80ul yeast competent cells, and

mixing the above linearized recombinant plasmids, transferring into 0.2cm electric transformation cup, and ice-cooling for 5 min;

(1-9-2) wiping water vapor, placing on an electric conversion instrument, and selecting yeast parameters for electric conversion;

(1-9-3) immediately add 1ml of ice-bath sorbitol and transfer the mixture to a 1.5ml EP tube;

(1-9-4) incubation at 30 ℃ with standing

(1-9-5) taking

The transformed bacterial solution was spread on YPD plates containing Zeocin (100ug/ml), and cultured by leaving to stand at 30 ℃

Observing the growth of the transformant;

(1-10) screening of Positive Strain

(1-10-1) selecting 20 clones from YPD culture plates of the transformed bacteria, respectively inoculating into 10ml of BMGY culture medium, and performing shake culture (225r/min) at 30 ℃ for 24 h;

(1-10-2) collecting 1ml of fermentation liquor for each clone, centrifuging (8000r/min) for 5min, and discarding the supernatant;

(1-10-3) adding 500ul of PBS buffer solution into the precipitate, and suspending the precipitate;

(1-10-4) centrifuging (8000r/min) for 3min, and discarding the supernatant;

(1-10-5) dissolving the precipitate with 100ul buffer TE, and carrying out boiling water bath for 10 min;

(1-10-6) -80 ℃ for 30 min;

(1-10-7) after boiling water bath for 10min again, centrifuging (5000r/min) for 5 min;

(1-10-8) collecting the supernatant, taking 10ul of the supernatant as a template for each clone, establishing a PCR reaction system (25 ul/tube), and using the product for yeast genome identification;

the PCR system is as follows:

①94℃2min；

② 30 cycles of 94 ℃ for 30s, 61 ℃ for 30s and 72 ℃ for 1 min;

③72℃10min；

carrying out 1% agarose gel electrophoresis on the amplification product, and observing whether a gene fragment with an expected size is obtained;

(1-11) inducible expression of transformants

(1-11-1) taking the clone strains with positive PCR identification results, respectively inoculating the clone strains into 10ml of BMGY (pH6.0) culture medium, performing shake culture at 30 ℃ for 24h, and collecting cells;

(1-11-2) resuspending cell precipitation of BMMY with the same volume (10ml), performing shake culture at 30 ℃, performing induced expression, supplementing methanol to the final concentration of 0.5% every 24h in the induction process, and supplementing DEPC water to keep the total volume of fermentation liquor unchanged;

(1-11-3) continuously inducing and culturing for 7d, taking 1ml fermentation liquor every 24h, centrifuging, and analyzing supernatant by SDS-PAGE of rhApoA-II and rhApoC-II.

Wherein, the step (2) comprises the following steps:

(2-1) culture of engineering bacteria

(2-1-1) streaking the frozen engineering bacteria on YPD agar plate at 30 deg.C

(2-1-2) until the colony grows to 2-3 mm, picking the monoclonal colony in 10ml YPD culture solution, and carrying out shake culture at 30 ℃ and 250r/min in a seed culture medium for 24 h;

(2-1-3) the above culture was added to 2L of YPD culture medium and subjected to shaking culture at 30 ℃ and 250r/min

(2-2) accumulation of Yeast cell Biomass

(2-2-1) preparing 40L FM21 culture medium, adding into 80L fermentation tank, sterilizing culture solution, fermentation tank and pipeline at 121 deg.C for 30min under high pressure;

(2-2-2) when the culture solution in the fermentation tank is cooled to room temperature, adjusting the pH value of the FM21 culture medium to a required value by using ammonia water, and then adding 48ml of PTM1 trace elements and 18ml of 0.2g/L biotin stock solution;

(2-2-3) inoculating the 2L YPD culture strain into a fermentation tank, starting fermentation culture, namely culturing and amplifying thallus by using glycerol in the first stage, wherein the parameters of the fermentation tank are respectively set to be a stirring speed of 600r/min, a pressure in the tank of 10psi and a temperature of 30 ℃, a dissolved oxygen value is set to be more than 20%, the control is carried out in a P-I-D mode, and pure oxygen is introduced according to needs;

(2-2-4) sampling for 1 time at intervals of 12h at the stage, measuring A600 and cell wet weight, analyzing the growth state of the yeast, observing the bacterial liquid under naked eyes and a mirror, confirming that no mixed bacteria pollution exists, and keeping the supernatant for later use;

(2-2-5) when the DO value rises to be close to 100% (about 24h), the glycerol in the culture solution is completely consumed, and the culture solution is shifted to a stage of supplementing the glycerol so as to further increase the cell density and accumulate the biomass;

(2-2-6) adding PTM1 trace element into 50% glycerol after autoclaving according to the proportion of adding 12ml PTM1 trace element into each liter of glycerol, uniformly mixing, adding into a fermentation tank at the speed of 728ml/h of initial fermentation liquid of 18.2ml/h/L until the thalli are wetHeavy goods

(2-2-7) after the glycerol is stopped being supplemented, observing that the DO value is increased to be close to 100%, continuously maintaining the starvation state of the glycerol for 30min, and transferring to a methanol induced expression stage;

(2-3) methanol-induced expression of rhApoA-II and rhApoC-II

(2-3-1) adding PTM1 microelement into methanol according to the proportion of 12ml/L, mixing uniformly, adding into a fermentation tank at the speed of 3.6ml/h/L initial fermentation liquid, namely 144ml/h for inducing expression, and maintaining the low speed

So as to adapt the yeast to an environment with methanol as the only carbon source; during the period, the DO value becomes unstable and fluctuates greatly, after the yeast adapts to the environment with methanol as the only carbon source, the DO value is kept stable, and the oxygen introduction amount is adjusted to maintain the DO at the constant

Continuously maintaining the low rate and supplementing methanol for 1 h;

(2-3-2) after the induction expression is started, sampling for 1 time at intervals of 6h, measuring the wet weight of A600 and cells, analyzing the growth state of the saccharomycetes, observing the bacterial liquid under the naked eye and the mirror, confirming that no mixed bacteria pollution exists, and reserving the supernatant for SDS-PAGE analysis;

(2-3-3) finishing fermentation after the induction fermentation is carried out for 60 hours;

(2-3-4) SDS-PAGE analysis of the change of rhApoA-II and rhApoC-II in the supernatant of fermentation liquor at different induction times.

Wherein, the step (3) comprises the following steps:

(3-1) centrifugal separation

Continuously centrifuging the fermentation liquor by a three-foot sedimentation centrifuge (2000r/min) and a tubular separator (15000r/min) to separate the supernatant of the fermentation liquor;

(3-2) SP Sepharose XL cation column chromatography

(3-2-1) determining the optimal binding pH and the optimal eluent (NaCl) concentration of rhApoA-II, rhApoC-II purified by SP Sepharose XL cation exchange chromatography through an AKTA explorer 100 rapid purification process development system;

(3-2-2) diluting the supernatant of the fermentation broth obtained by centrifugation with 3 times the volume of water and adding 1/10 volumes of solution A, adjusting the pH to the above-mentioned optimum binding pH with HCl;

(3-2-3) equilibrating the SP Sepharose XL cation chromatography column with 10 column volumes of solution B (pH at the optimum value optimized above);

(3-2-4) adding the sample obtained in the step (3-2-2) into a balanced SP Sepharose XL cation chromatographic column, and monitoring at the wavelength of 280 nm;

(3-2-5) stopping sample adding when rhApoA-II and rhApoC-II appear in the penetrating fluid, and flushing the resin with solution B until the A280 value is reduced to a baseline;

(3-2-6) eluting with the solution E, and collecting protein peaks step by step;

(3-3) Source 30RPC reversed-phase hydrophobic column chromatography

(3-3-1) equilibrating a Source 30RPC reverse-phase hydrophobic chromatography column with 10 column volumes of solution G;

(3-3-2) loading the collected eluted protein sample on a balanced Source 30RPC reverse-phase hydrophobic chromatography column, and monitoring under a wave K280 nm;

(3-3-3) stopping sample adding when rhApoA-II and rhApoC-II appear in the penetrating fluid, and flushing the resin with solution G until the A280 value is reduced to a baseline;

(3-3-4) eluting with solution H, and collecting protein peaks step by step;

(3-4) distillation under reduced pressure

Distilling the methanol eluate at 37 deg.C under reduced pressure in a lifting constant temperature bath, and removing methanol and concentrating.

Wherein, the detailed steps of the step (4) are as follows: the purity and concentration of the purified ApoA-II and ApoC-II are identified by SDS-PAGE analysis, and the lipoprotein concentration is quantified by immunoturbidimetry and Bradford method; and (3) diluting the lipoprotein with the known concentration by using human derived hormone-removed serum, determining the content of the sample to be detected by using an IFCC-WHO lipoprotein standard substance as a tracing source through immunoturbidimetry, then diluting the sample to 2 levels by using the hormone-removed serum according to the determined value level, and subpackaging to construct a freeze-dried powder quality control commodity.

The technical scheme of the invention has the following beneficial effects: the ApoA-II and ApoC-II detection mixed quality control product prepared by using the eukaryotic cell system has the advantages of good stability, low cost, simple and convenient use, contribution to animal experiments, no influence by transportation and temperature factors, good detection repeatability and high stability, can meet the quality control requirements of clinic on ApoA-II and ApoC-II detection, and can improve the accuracy of clinical sample detection results.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

The invention provides an ApoA-II and ApoC-II detection mixed quality control product which is a freeze-dried product of trihydroxymethyl aminomethane, polyethylene glycol 6000, ProClin 950, deexcitin serum and two protein positive substances, namely ApoA-II and ApoC-II.

The specific components are shown in the following table:

note: beating into main raw materials.

(1) preparing human ApoA-II and ApoC-II gene clone and expression vector, which comprises the following steps:

poA-II-F：ATGAAGCTGCTCCAGCAACTGT；

ApoA-II-R：TTATCACTGGGTGGCAGGCTGT；

ApoC-II-F：TCCAGCAGCAAGATTCAGAGTGCC；

ApoC-II-R：TGGGATGTCACCCTTCAGGGTC；

the reaction system is as follows:

amplification was according to the following PCR amplification procedure:

①94℃2min；

② 30 cycles of 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 30 sec;

③72℃10min；

(1-4)XL₁preparation of Blue competent cells

(1-4-2) centrifuging at 4 ℃ for 10min (4000r/min) and collecting cells;

(1-4-4) with 60ml of precooled 0.1mol/L CaCl₂-MgCl₂Resuspending the cells;

(1-4-5) centrifuging at 4 ℃ (4000r/min) for 10min, and recovering cells;

(1-5) transformation of recombinant plasmid

(1-5-2) carrying out ice bath for 30 min;

(1-6) construction of rhApoA-I Pichia pastoris secretory expression vector

an upstream primer: 31bp

5’-AAACTCGAGAAGAGAGATGAACCCCCCCAGA-3’；

A downstream primer: 32bp

5’-GCGTCTAGATCACTGGGTGTTGAGCTTCTTAG-3’；

(1-6-2) PCR reaction

①94℃ 4min；

② 30 cycles of 94 ℃ for 30s, 61 ℃ for 30s and 72 ℃ for 1 min;

③72℃ 10min；

(1-8) preparation of Yeast competence

When the colony grows to 2-3 mm, selecting a single colony, inoculating the single colony in 5ml of YPD culture medium, and performing shake culture at 225r/min and 30 ℃ for 24 hours;

(1-8-3) centrifuging at 4 ℃ (3000r/min) for 5min, and collecting cells;

(1-9) transformation

(1-9-1) taking 80ul yeast competent cells, and

(1-9-4) incubation at 30 ℃ with standing

(1-9-5) taking

Observing the growth of the transformant;

(1-10) screening of Positive Strain

(1-10-4) centrifuging (8000r/min) for 3min, and discarding the supernatant;

(1-10-6) -80 ℃ for 30 min;

the PCR system is as follows:

①94℃ 2min；

② 30 cycles of 94 ℃ for 30s, 61 ℃ for 30s and 72 ℃ for 1 min;

③72℃ 10min；

(1-11) inducible expression of transformants

(2) The method comprises the following steps of utilizing an 80L fermentation tank to carry out large-scale fermentation expression on rhApoA-II and rhApoC-II:

(2-1) culture of engineering bacteria

(2-1-1) streaking the frozen engineering bacteria on YPD agar plate at 30 deg.C

(2-1-2) until the colony grows to about 2mm, picking the monoclonal colony in 10ml YPD culture solution and seed culture medium, and carrying out shaking culture at 30 ℃ and 250r/min for 24 h;

(2-2) accumulation of Yeast cell Biomass

(2-2-6) adding PTM1 trace element into 50% glycerol after autoclaving according to the proportion of adding 12ml PTM1 trace element into each liter of glycerol, uniformly mixing, adding into a fermentation tank at the speed of 728ml/h of initial fermentation liquid of 18.2ml/h/L until the wet weight of thalli reaches

(2-3) methanol-induced expression of rhApoA-II and rhApoC-II

Continuously maintaining the low rate and supplementing methanol for 1 h;

(3) The purification of rhApoA-II and rhApoC-II comprises the following steps:

(3-1) centrifugal separation

(3-2) SP Sepharose XL cation column chromatography

(3-2-6) eluting with the solution E, and collecting protein peaks step by step;

(3-3) Source 30RPC reversed-phase hydrophobic column chromatography

(3-3-4) eluting with solution H, and collecting protein peaks step by step;

(3-4) distillation under reduced pressure: distilling the methanol eluate at 37 deg.C under reduced pressure in a lifting constant temperature bath, and removing methanol and concentrating.

(4) Preparing, assigning and packaging ApoA-II and ApoC-II protein quality control products: the purity and concentration of the purified ApoA-II and ApoC-II are identified by SDS-PAGE analysis, and the lipoprotein concentration is quantified by immunoturbidimetry and Bradford method; and (3) diluting the lipoprotein with the known concentration by using human derived hormone-removed serum, determining the content of the sample to be detected by using an IFCC-WHO lipoprotein standard substance as a tracing source through immunoturbidimetry, then diluting the sample to 2 levels by using the hormone-removed serum according to the determined value level, and subpackaging to construct a freeze-dried powder quality control commodity.

The invention also provides a test of the ApoA-II and ApoC-II detection mixed quality control product, which comprises uniformity detection, stability detection and transportation stability detection, and specifically comprises the following steps:

1. uniformity detection

Taking 10 bottles of each horizontal quality control product of the same batch number, testing each bottle of quality control product for 1 time by using corresponding reagent, and calculating the average value of 10 test results of each horizontal time

And standard deviation S₁(ii) a One of the bottles was tested 10 times per level and the average of the 10 test results was calculated

And standard deviation S₂(ii) a The variation coefficient (CV%) between bottles is calculated according to the following formula and should not be more than 10.0%.

Equation 1:

equation 2:

equation 3:

equation 4:

when S1 is less than S2, let CV bottle be 0

In the formula:

-mean value;

s-standard deviation;

n- — number of measurements;

X_i-designateThe ith measurement of the parameter.

Through actual detection and calculation, the bottle-to-bottle variation of the ApoA-II and ApoC-II detection mixed quality control product prepared by the method is less than 10.0 percent, and the uniformity requirement is met.

2. Stability detection

2.1 reconstitution stability

After three batches of freeze-dried quality control products are redissolved, the freeze-dried quality control products are placed at the temperature of 2-8 ℃, are respectively examined for 14 days, 10 days and 7 days after redissolution, are placed in reverse order, and are expired in the same day at all time points, and are detected by using corresponding kits.

And (4) conclusion: all results are within the required range, meeting the requirements of expected results.

2.2 stability of acceleration

The main basis is as follows: accelerated stability, i.e., transient conditions are used to increase the rate of chemical or physical degradation of the product, thereby predicting its useful life. According to the arrhenius formula: the storage period of the finished product at 2-8 ℃ is 6 months, and the finished product is examined for 3 days at 37 ℃; the storage period of the finished product at 2-8 ℃ is 12 months, and the finished product is examined for 7 days in a 37 ℃ environment; the storage period of the finished product at 2-8 ℃ is 18 months and the finished product is examined for 10 days in the environment of 37 ℃; the storage period of the finished product at 2-8 ℃ is 24 months, and the finished product is examined for 14 days at 37 ℃.

The method comprises the following steps: the three batches of freeze-dried quality control products are examined for 14 days, 10 days, 7 days and 3 days respectively at the temperature of 37 ℃, placed in a reverse order, expired at the same day at all time points, and detected by using corresponding kits.

And (4) conclusion: the three batches of quality control materials are tested at 37 ℃ for 14 days in accelerated stability, and the test results of each level are in a given range, thereby meeting the requirements of expected results.

2.3 stability in transportation

In order to ensure the effectiveness of a quality control product in the using process of a user, the quality control product is simulated and transported for 7 days at 37 ℃, and then stored according to a storage mode (2 ℃ -8 ℃) expected and specified by the quality control product, the assessment principle is that the performance of the quality control product two months after the expiration date, such as the quality control product stored for 2 years at 2 ℃ -8 ℃, is recommended to be detected at 0 th, 4 th, 8 th, 12 th, 16 th, 24 th and 26 th months respectively, and the effectiveness of the inflammation quality control product after long-term storage is ensured.

And (4) conclusion: three batches of quality control products are simulated and transported at 37 ℃ for 7 days, and then are placed in real time for 26 months without obvious abnormality, so that the natural hollow products can be transported in shade for 6 days, and the effective period can reach 24 months.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Scorzon Biotechnology Ltd

<120> ApoA-II and ApoC-II detection mixed quality control product and preparation method thereof

<130> 2020-9-26

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 22

<212> DNA

<213> unknown

<400> 1

atgaagctgc tccagcaact gt 22

<210> 2

<211> 22

<212> DNA

<213> unknown

<400> 2

ttatcactgg gtggcaggct gt 22

<210> 3

<211> 24

<212> DNA

<213> unknown

<400> 3

tccagcagca agattcagag tgcc 24

<210> 4

<211> 22

<212> DNA

<213> unknown

<400> 4

tgggatgtca cccttcaggg tc 22

<210> 5

<211> 31

<212> DNA

<213> unknown

<400> 5

aaactcgaga agagagatga acccccccag a 31

<210> 6

<211> 32

<212> DNA

<213> unknown

<400> 6

gcgtctagat cactgggtgt tgagcttctt ag 32

Claims

1. An ApoA-II and ApoC-II detection mixed quality control product is characterized by being a freeze-dried product comprising two protein positive substances, namely deexcitin serum and ApoA-II and ApoC-II.

2. The ApoA-II, ApoC-II assay mixed quality control product of claim 1 further comprising tris, peg 6000 and ProClin 950.

3. The ApoA-II and ApoC-II assay mixed quality control product of claim 2, wherein the concentration of tris is 99.9% or more.

4. A method of preparing an ApoA-II, ApoC-II assay mixed quality control according to any one of claims 1 to 3, comprising the steps of:

(1) preparing human ApoA-II and ApoC-II gene clone and expression vector;

(3) purifying rhApoA-II and rhApoC-II;

5. The method for preparing ApoA-II and ApoC-II mixed quality control substance for detection according to claim 4, wherein the step (1) comprises the following steps:

grinding 300mg of frozen tissue, transferring the ground tissue into a 50ml centrifuge tube, adding 5ml Trizol, homogenizing at high speed for 15-30sec at room temperature by using a homogenizer, adding 1.0ml chloroform, shaking up sufficiently, standing at room temperature for 5min, and centrifuging at 4 ℃ for 15 min;

transferring the upper layer water phase into another centrifuge tube, adding isopropanol with the same volume, shaking up, precipitating for 10min, centrifuging at 4 deg.C for 15min, discarding supernatant, adding 1ml of 75% ethanol, washing precipitate, centrifuging at 4 deg.C for 5min, dissolving total RNA precipitate in 50ul DEPC treated water, and storing at-80 deg.C for use;

poA-II-F：ATGAAGCTGCTCCAGCAACTGT；

ApoA-II-R：TTATCACTGGGTGGCAGGCTGT；

ApoC-II-F：TCCAGCAGCAAGATTCAGAGTGCC；

ApoC-II-R：TGGGATGTCACCCTTCAGGGTC；

the reaction system is as follows:

amplification was according to the following PCR amplification procedure:

①94℃2min；

② 30 cycles of 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 30 sec;

③72℃10min；

(1-4)XL₁preparation of Blue competent cells

(1-4-2) centrifuging at 4 ℃ for 10min and collecting cells;

(1-4-4) with 60ml of precooled 0.1mol/L CaCl₂-MgCl₂Resuspending the cells;

(1-4-5) centrifuging at 4 ℃ for 10min, and recovering cells;

(1-5) transformation of recombinant plasmid

(1-5-2) carrying out ice bath for 30 min;

(1-5-4) adding 800ul of preheated SOC culture medium into the tube, transferring the tube to a shaking table for shake culture for 1h, so that bacteria are revived and the antibiotic resistance marker gene coded by the plasmid is expressed;

(1-5-5) spreading 200ul of transformed competent cells on an LB plate culture medium containing Amp, standing at room temperature until liquid is absorbed, and performing inverted culture at 37 ℃ for 12-16 h;

(1-6) construction of rhApoA-I Pichia pastoris secretory expression vector

(1-6-1) designing primers according to hApoA-IDNA sequences published by GenBank, and amplifying ApoA-II and ApoC-II coding sequences of which the 5 'end introduces Xhol restriction endonuclease sites and Kex2 protease recognition sequence coding genes and the 3' end introduces Xbal restriction endonuclease sites;

an upstream primer: 31bp

5’-AAACTCGAGAAGAGAGATGAACCCCCCCAGA-3’；

A downstream primer: 32bp

5’-GCGTCTAGATCACTGGGTGTTGAGCTTCTTAG-3’；

(1-6-2) PCR reaction

①94℃4min；

② 30 cycles of 94 ℃ for 30s, 61 ℃ for 30s and 72 ℃ for 1 min;

③72℃10min；

(1-8) preparation of Yeast competence

(1-8-1) taking a pichia pastoris X-33 strain, carrying out streak culture on a YPD negative culture plate without antibiotics, and carrying out 30 ℃ for 2-3 d; when the colony grows to 2-3 mm, selecting a single colony, inoculating the single colony in 5ml YPD medium, and performing shake culture at 225r/min and 30 ℃ for 24 h;

(1-8-3) centrifuging at 4 ℃ for 5min, and collecting cells;

(1-8-6) resuspending the cells with 20ml of pre-cooled 1mol/L D-sorbitol, centrifuging at 4 ℃ for 5min, and discarding the supernatant;

(1-9) transformation

(1-9-1) mixing 80ul yeast competent cells with 10-15 ul of the linearized recombinant plasmid, transferring into a 0.2cm electrotransformation cup, and carrying out ice bath for 5 min;

(1-9-4) standing and incubating for lh-2 h at 30 ℃;

(1-9-5) coating 100-200 ul of transformed bacterial liquid on a YPD plate containing Zeocin, standing and culturing at 30 ℃ for 2-3 d, and observing the growth of a transformant;

(1-10) screening of Positive Strain

(1-10-1) selecting 20 clones from YPD culture plates of the transformed bacteria, respectively inoculating the clones into 10ml of BMGY culture medium, and performing shake culture at 30 ℃ for 24 hours;

(1-10-2) collecting 1ml of fermentation liquor for each clone, centrifuging for 5min, and removing supernatant;

(1-10-4) centrifuging for 3min, and removing the supernatant;

(1-10-5) dissolving the precipitate with 100ul bufferTE, and carrying out boiling water bath for 10 min;

(1-10-6) -80 ℃ for 30 min;

(1-10-7) centrifuging for 5min after boiling water bath for 10min again;

(1-10-8) collecting the supernatant, taking 10ul of the supernatant as a template for each clone, establishing a PCR reaction system, and using the product for yeast genome identification;

the PCR system is as follows:

①94℃2min；

② 30 cycles of 94 ℃ for 30s, 61 ℃ for 30s and 72 ℃ for 1 min;

③72℃10min；

(1-11) inducible expression of transformants

(1-11-1) taking the clone strains with positive PCR identification results, respectively inoculating the clone strains into 10ml of BMGY culture medium, performing shake culture at 30 ℃ for 24h, and collecting cells;

(1-11-2) resuspending cell precipitation of BMMY with the same volume, performing shake culture at 30 ℃, performing induced expression, supplementing methanol to the final concentration of 0.5% every 24h in the induction process, and supplementing DEPC water to keep the total volume of the fermentation liquor unchanged;

6. The method for preparing ApoA-II and ApoC-II mixed quality control substance for detection according to claim 4, wherein the step (2) comprises the following steps:

(2-1) culture of engineering bacteria

(2-1-1) carrying out streak culture on the frozen engineering bacteria on an YPD agar plate at 30 ℃ for 2-3 d;

(2-1-3) adding the culture into a 2LYPD culture solution, and carrying out shaking culture at 30 ℃ and 250r/min for 16-24 h;

(2-2) accumulation of Yeast cell Biomass

(2-2-1) preparing a 40LFM21 culture medium, adding the culture medium into an 80L fermentation tank, sterilizing the culture medium, the fermentation tank and a pipeline at 121 ℃ for 30min under high pressure;

(2-2-3) inoculating the 2LYPD culture strain into a fermentation tank, starting fermentation culture, wherein the first stage is glycerol culture and thallus amplification, the parameters of the fermentation tank are respectively set as the stirring speed of 600r/min, the pressure in the tank is 10psi, the temperature is 30 ℃, the dissolved oxygen value is set to be more than 20%, the control is carried out in a P-I-D mode, and pure oxygen is introduced according to the requirement;

(2-2-5) when the DO value rises to be close to 100%, which indicates that the glycerol in the culture solution is completely consumed, shifting to a stage of supplementing the glycerol so as to further increase the cell density and accumulate the biomass;

(2-2-6) adding PTM1 trace element into 50% autoclaved glycerol according to the proportion of adding 12ml of PTM1 trace element into each liter of glycerol, uniformly mixing, and adding into a fermentation tank at the speed of 728ml/h of initial fermentation liquid of 18.2ml/h/L until the wet weight of thalli reaches 180-220 g/L;

(2-3) methanol-induced expression of rhApoA-II and rhApoC-II

(2-3-1) adding PTM1 trace element into methanol according to the proportion of 12ml/L, uniformly mixing, adding the mixture into a fermentation tank at the speed of 3.6ml/h/L of initial fermentation liquid, namely 144ml/h, for inducing expression, and maintaining the low speed for 2 h-3 h so as to enable the yeast to adapt to the environment with methanol as the only carbon source; during the period, the DO value becomes unstable and fluctuates greatly, after the yeast adapts to the environment taking methanol as the only carbon source, the DO value is kept stable, the oxygen introduction amount is adjusted to maintain the DO at 20-30 ℃, and the low-speed methanol supplementation is continuously maintained for 1 hour;

7. The method for preparing ApoA-II and ApoC-II mixed quality control substance for detection according to claim 4, wherein the step (3) comprises the following steps:

(3-1) centrifugal separation

Continuously centrifuging the fermentation liquor by a three-foot sedimentation centrifuge and a tubular separator to separate the supernatant of the fermentation liquor;

(3-2) SP Sepharose XL cation column chromatography

(3-2-1) determining the optimal binding pH and the optimal eluent concentration of rhApoA-II and rhApoC-II purified by SP Sepharose XL cation exchange chromatography through an AKTA explorer 100 rapid purification process development system;

(3-2-3) equilibrating the SP Sepharose XL cation chromatography column with 10 column volumes of solution B;

(3-2-6) eluting with the solution E, and collecting protein peaks step by step;

(3-3) Source 30RPC reversed-phase hydrophobic column chromatography

(3-3-4) eluting with solution H, and collecting protein peaks step by step;

(3-4) distillation under reduced pressure

8. The method for preparing ApoA-II and ApoC-II mixed quality control substance for detection according to claim 4, wherein the detailed step of step (4) is: the purity and concentration of the purified ApoA-II and ApoC-II are identified by SDS-PAGE analysis, and the lipoprotein concentration is quantified by immunoturbidimetry and Bradford method; and (3) diluting the lipoprotein with the known concentration by using human derived hormone-removed serum, determining the content of the sample to be detected by using an IFCC-WHO lipoprotein standard substance as a tracing source through immunoturbidimetry, then diluting the sample to 2 levels by using the hormone-removed serum according to the determined value level, and subpackaging to construct a freeze-dried powder quality control commodity.