Recombinant human lipoprotein and preparation method of polyclonal antibody thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a recombinant human lipoprotein a and a preparation method of a polyclonal antibody thereof, which comprises the following steps: by taking pET28a (+) as an expression vector, introducing an Lp (a) gene of which the N-terminal is fused with six His tags, Trx tags and PP enzyme digestion sites under the control of a T7 promoter into escherichia coli for induction, denaturating and renaturating an inclusion body, purifying by a Ni Focurose 6FF column, and purifying by using a recombinant human lipoprotein a polyclonal antibody. The purity of the Lp (a) fusion protein is greater than or equal to 90%; the Lp (a) fusion protein is subjected to enzyme digestion and passes through a Ni Focurose 6FF column for purification, and the label-free Lp (a) protein with the purity larger than or equal to 95% is obtained; the Lp (a) polyclonal antibody is obtained by immunizing a goat with Lp (a) protein with the purity greater than or equal to 95%, collecting serum after each immunization, screening qualified serum through an antiserum bidirectional agar diffusion experiment, and purifying with Protein A. The Lp (a) prepared by the method is low in cost and relatively high in purity, and can be used as an effective immunogen for preparing the Lp (a) polyclonal antibody; and the material can be used as a calibrator raw material of a detection kit.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a method for preparing recombinant human lipoprotein A and a polyclonal antibody thereof. Background Art

[0002] Lp(a) is composed of a lipid core, apolipoprotein A (ApoA) and ApoB100. The lipid core is similar to LDL-C, and ApoA is similar to plasminogen in structure. Therefore, Lp(a) may have atherogenic and thrombogenic effects. Lp(a) is more likely to cause atherosclerosis than LDL and has a longer plasma residence time. The level of circulating Lp(a) is mainly determined by the LPA gene encoding ApoA and is not affected by age, gender, exercise, diet and nutrition. Epidemiological studies have shown that increased Lp(a) concentrations are associated with an increased risk of coronary heart disease (CHD).

[0003] Patients with elevated LP(a) levels have a 2-3 times higher risk of cardiovascular disease than normal people. Elevated lipoprotein a can easily lead to atherosclerosis, forming atherosclerotic plaques, leading to vascular stenosis and affecting the blood supply to the cardiovascular and cerebrovascular systems. In this case, it is easy to induce myocardial infarction, myocardial ischemia, coronary artery insufficiency, coronary heart disease, as well as cerebral vascular stenosis, carotid artery stenosis and other diseases. In addition, LP(a) may also increase the risk of cerebral thrombosis and cerebral infarction, thereby causing stroke. Like other blood lipids, LP(a) levels are mainly determined by genes (>90%) and cannot be changed by traditional lifestyle interventions and statin therapy. Therefore, LP(a) is regarded as a more vicious "bad" cholesterol than low-density lipoprotein cholesterol (LDL-C), and has also been shown to be an independent risk factor for cardiovascular disease such as atherosclerosis and aortic valve stenosis.

[0004] The detection of Lp(a) requires highly pure Lp(a) antigen and high-performance Lp(a) antibody. At present, Lp(a) is mainly obtained through natural extraction and genetic engineering. Because natural Lp(a) is difficult to obtain and expensive, and it is difficult to remove cholesterol, TG and apolipoprotein B during the preparation of antigens, people are increasingly favoring the synthesis of active recombinant Lp(a) through genetic engineering, which not only avoids the limitation of raw material sources, but also reduces costs and can be mass-produced. High-quality Lp(a) immunogens are the key to the preparation of high-quality Lp(a) antibodies. At present, most Lp(a) detection laboratories use immunological methods. Commonly used detection methods include immunoturbidimetry (ITA), immunoturbidimetry (INA), fluorescence test and enzyme-linked immunosorbent assay (ELISA), which require Lp(a) antibodies with good specificity and sensitivity. In order to enable many patients to receive good detection and treatment, the development of low-cost, active, high-purity Lp(a) and high-performance Lp(a) polyclonal antibodies has great economic value and positive practical significance. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing recombinant human lipoprotein a and its polyclonal antibody, aiming to produce low-cost, active, high-purity Lp(a) and its high-performance Lp(a) polyclonal antibody.

[0006] To achieve the above object, the present invention provides a method for preparing recombinant human lipoprotein a and its polyclonal antibody, comprising the following steps:

[0007] Using pET28a(+) as the expression vector, the Lp(a) gene with 6 His tags, Trx tags and PPase cleavage sites fused to the N-terminus under the control of T7 promoter was introduced into Escherichia coli for induction, and then the inclusion bodies were denatured and renatured and purified by NiFocurose 6FF column to obtain Lp(a) fusion protein with a purity of ≥90%;

[0008] The Lp(a) fusion protein was cleaved and purified by Ni Focurose 6FF column to obtain Lp(a) protein without tag with a purity of ≥95%;

[0009] After goats were immunized with Lp(a) protein with a purity of ≥95%, serum was collected after each immunization, and qualified serum was screened by antiserum two-way agar diffusion experiment, and purified using Protein A to obtain Lp(a) polyclonal antibodies.

[0010] Wherein, the Escherichia coli is Ecoli BL21 (DE3).

[0011] The E. coli fermentation conditions are as follows: the seed liquid is inoculated into 1000 ml fermentation medium containing 100 ug / mL kan at a 5% inoculation rate, and the culture is expanded at 37°C and 220 rpm for about 1-2 hours. When the OD600 rises to 0.8, IPTG is added to make the final concentration of IPTG in the fermentation liquid 0.5 mM / L, and the culture is continued in a shaker at 18°C ​​and 220 rpm for 12-14 hours.

[0012] The fermentation medium contains 15 g / L tryptone, 25 g / L yeast powder, 0.6 g / L MgSO4, 23 g / L Na2HPO4, 5 g / L NaH2PO4 and 15 g / L glucose, and the pH is 7.2.

[0013] The invention discloses a method for preparing a recombinant human lipoprotein a and a polyclonal antibody thereof. The method comprises the following steps: using pET28a(+) as an expression vector, introducing an Lp(a) gene fused with 6 His tags, a Trx tag and a PP enzyme cleavage site at the N-terminus under the control of a T7 promoter into Escherichia coli for induction, and then subjecting the inclusion body to denaturation and renaturation and then purifying the Lp(a) fusion protein with a purity of ≥90% by using a Ni Focurose 6FF column; cleaving the Lp(a) fusion protein and purifying the Lp(a) protein with a purity of ≥95% by using a Ni Focurose 6FF column; immunizing goats with the Lp(a) protein with a purity of ≥95%, collecting serum after each immunization, screening qualified serum through an antiserum two-way agar diffusion experiment, and purifying the serum using Protein A to obtain an Lp(a) polyclonal antibody. In BL21(DE3), pET23a(+) was used as an expression vector, and the apoaKIV1(20-130) segment was selected for expression. The gene fragments with restriction enzyme cutting sites BamHI and EcoRI were inserted at both ends to achieve Lp(a) fusion protein inclusion body expression. The inclusion body was denatured and renatured, and then purified by Ni Focurose 6FF column to obtain Lp(a) fusion protein with a purity of ≥90%. The Lp(a) fusion protein was enzymatically cleaved and purified by Ni Focurose 6FF column to obtain Lp(a) protein without tag with a purity of ≥95%. The high-purity recombinant Lp(a) of the present invention is not only active, but also low in cost, not limited by raw materials, can be mass-produced, and has potential and wide application value in clinical diagnosis and in vitro diagnosis. Goats are immunized with lipoprotein(a) multiple times, blood is collected after each immunization, qualified serum is screened by two-way agar diffusion experiment, and purified by antigen affinity purification column to obtain highly active Lp(a) polyclonal antibodies. The prepared Lp(a) has low cost, simple operation, and high purity. It can not only be used as an effective immunogen, but also can be used for the preparation of Lp(a) polyclonal antibodies; and can also be used as a calibration material for a detection kit. The antibody prepared by the present invention has good specificity (high analytical sensitivity), a wide linear range, and a low detection limit, and has potential and wide application value in clinical diagnosis and in vitro diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1The present invention provides a flow chart of a method for preparing recombinant human lipoprotein a and its polyclonal antibody.

[0016] Figure 2 This is a purity test chart of high-purity sheep anti-human Lp(a) polyclonal antibody.

[0017] Figure 3 It is a linear graph of the kit of the present invention and the control kit. DETAILED DESCRIPTION

[0018] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0019] See also Figures 1 to 3 The present invention provides a method for preparing recombinant human lipoprotein a and its polyclonal antibody, comprising the following steps:

[0020] S1 used pET28a(+) as the expression vector, and introduced the Lp(a) gene with 6 His tags, Trx tags and PPase cleavage sites fused to the N-terminus under the control of T7 promoter into Escherichia coli for induction, and then denatured and renatured the inclusion bodies and purified them with NiFocurose 6FF column to obtain Lp(a) fusion protein with a purity of ≥90%;

[0021] In an embodiment of the present invention, the Escherichia coli is Ecoli BL21 (DE3), and the fermentation conditions of the Escherichia coli are as follows: the seed liquid is inoculated into 1000 ml fermentation medium containing 100 ug / mL kan at a 5% inoculation amount, and the culture is expanded at 37° C. and 220 rpm for about 1-2 hours. When OD600 rises to 0.8, IPTG is added to make the final concentration of IPTG in the fermentation broth 0.5 mM / L, and the fermentation broth is placed in a shaker at 18° C. and 220 rpm and cultured for 12-14 hours. The fermentation medium contains 15 g / L of tryptone, 25 g / L of yeast powder, 0.6 g / L of MgSO4, 23 g / L of Na2HPO4, 5 g / L of NaH2PO4 and 15 g / L of glucose, and the pH is 7.2.

[0022] S2: cleavage of Lp(a) fusion proteinase and purification by Ni Focurose 6FF column to obtain Lp(a) protein without tag with a purity of ≥95%;

[0023] S3 immunizes goats with Lp(a) protein with a purity of ≥95%, collects serum after each immunization, screens qualified serum through antiserum two-way agar diffusion experiment, and purifies it using Protein A to obtain Lp(a) polyclonal antibodies.

[0024] In the embodiment of the present invention, goats were immunized with lipoprotein (a) multiple times, blood was collected after each immunization, qualified serum was screened by two-way agar diffusion experiment, and purified by antigen affinity purification column to obtain highly active Lp(a) polyclonal antibodies.

[0025] In order to better understand the present technical solution, the following embodiments are provided for further explanation:

[0026] Example 1 Fermentation of recombinant Lp(a) expression strain

[0027] The BL21 (pET23a-apoaKIV1 (20-130)) recombinant strain with good activity was taken out from the -20°C refrigerator, dissolved, 50uL of the bacterial solution was added to 5ml LB medium containing 100ug / mL AMP, mixed, and cultured at 37°C, 220rpm for 12-14 hours. Transfer 5 mL of the cultured seed solution to 50 ml of LB medium containing 100 ug / mL AMP, and expand the culture at 37°C and 220 rpm for about 2-4 hours. When OD600 rises to about 1.0, transfer 50 mL of the cultured seed solution to 1000 ml of fermentation medium containing 100 ug / mL AMP, and expand the culture at 37°C and 220 rpm for about 1-2 hours. When OD600 rises to 0.8, add IPTG to make the final concentration of IPTG in the fermentation liquid 0.5 mM / L, and continue to culture in a shaker at 18°C ​​and 220 rpm for 12-14 hours.

[0028] Example 2 Collection and lysis of bacteria

[0029] After the fermentation is completed, centrifuge the bacterial solution at 4℃5000rpm for 15min, discard the supernatant and collect the bacteria. Wash the bacteria with an equal volume of cold PBS buffer, then centrifuge at 4℃6000rpm for 15min to collect the bacteria. Resuspend the bacteria in PBS with 1 / 20 of the volume of the fermentation liquid, and place it in an ice-water mixture for ultrasonic cell wall breaking. The working parameters of the ultrasonic instrument are: ultrasonic power 75%, ultrasonic 5s, interval 10s, total time 35min, alarm temperature 20℃. Centrifuge the bacterial lysate at 4℃9000rpm for 30min, collect the precipitate, and use it for denaturation and renaturation of Lp(a) fusion protein.

[0030] Example 3 Inclusion body denaturation and renaturation

[0031] Wash the precipitate with 1 / 2 fermentation broth volume of precipitation washing solution, centrifuge at 4℃9000rpm for 30min, and collect the precipitate. Add 1 / 4 fermentation broth volume of inclusion body denaturation solution to resuspend the precipitate, turn it upside down for 30min, centrifuge at 4℃9000rpm for 30min, and collect the supernatant. Put it into a dialysis bag (molecular weight cutoff 10KDa) and tie it up, then immerse the dialysis bag in 5L 4℃ inclusion body refolding solution ① for dialysis, change to inclusion body refolding solution ② after 12-14h and continue dialysis for 12-14h, then change to inclusion body refolding solution ③ and continue dialysis for 12-14h. Centrifuge the solution in the dialysis bag, centrifuge at 4℃9000rpm for 30min, collect the supernatant, and you can get a soluble Lp(a) fusion protein crude extract.

[0032] Example 4 Lp(a) fusion protein purification

[0033] The supernatant of the denatured and refolded Lp(a) fusion protein was first purified by nickel ion affinity chromatography, and the NiFocurose 6FF filler was loaded into the column, washed with 10 column volumes of deionized water, and then balanced with 10 column volumes of Ni Focurose 6FF balance solution. After the balance was completed, the supernatant of the lysate was passed through the column. After the column was completed, the impurities were washed with 10 column volumes of Ni Focurose 6FF washing solution. Finally, 1.5-3 volumes of Ni Focurose 6FF eluent were added for elution, and the washing solution with the peak was collected (detected with a protein detector). The Ni Focurose 6FF eluent was the Lp(a) fusion protein.

[0034] Example 5 Lp(a) fusion protein dialysis and enzyme digestion

[0035] The purified eluate was placed in a dialysis bag (molecular weight cutoff 10KDa), tied, and then immersed in 5L 4℃ Ni Focurose 6FF equilibrium solution for dialysis. The Ni Focurose 6FF equilibrium solution was changed every 12 hours, and the Ni Focurose 6FF equilibrium solution was changed three times in total to remove the imidazole in the eluate. 1U of 3C enzyme was added for every 20mg of Lp(a) fusion protein, mixed, and digested at 4℃ for 14-16h.

[0036] Example 6 Lp(a) purification

[0037] The purification adopts the method of nickel ion affinity chromatography purification. After the filler is loaded into the column, it is first washed with 10 column volumes of deionized water, and then balanced with 10 column volumes of Ni Focurose 6FF balance solution. After the balance is completed, the fusion proteinase cleavage solution is passed through the column. After the column is completed, the impurities are washed with 10 column volumes of Ni Focurose 6FF washing solution. Finally, 1.5-3 volumes of NiFocurose 6FF eluent are added for elution, and the washing solution with peaks is collected (detected with a protein detector). The Ni Focurose 6FF eluent is the Lp(a) protein.

[0038] Example 7: Purification of Lp(a) polyclonal antibodies

[0039] The β2-microglobulin prepared above was used to immunize sheep multiple times, and blood was collected after each immunization. Qualified serum was screened by two-way agar diffusion experiment. Lp(a) polyclonal antibody was purified by antigen affinity purification. The collected antiserum was centrifuged at 4℃9000rpm for 20min, filtered to remove the precipitate, and mixed with an equal volume of binding buffer. ProteinA was balanced with 5 column volumes of binding buffer, and the sample was loaded according to the volume of 1-5mL antiserum corresponding to 1ml filler, and the loading was repeated twice. After the column was completed, the impurities were washed with 5 column volumes of binding buffer. Finally, the elution buffer was used for elution, and the washing liquid with the peak was collected (detected by protein detector). At the same time, Tris-HCl (pH8-9) was used to adjust the pH of the eluate to keep it neutral. The eluate was the Lp(a) polyclonal antibody.

[0040] Add (NH4)2SO4 to the eluate to make its saturation 40%-60%, stirring while adding, and stir for 0.5h after all the solids are dissolved, and let it stand for 4h. After centrifugation at 4℃9000rpm for 20min, remove the supernatant, and use PBS buffer 1 (pH7-8) to redissolve the precipitate. Control the total protein concentration of the reconstituted solution to 10-30mg / mL. Load the reconstituted solution into a dialysis bag (molecular weight cutoff 7-14KDa), clamp the dialysis clamp, and then immerse the dialysis bag in PBS buffer for 48h, changing the buffer every 6-10h. After centrifugation, the protein solution is filtered through a 0.22μm filter membrane, and 0.01-0.5% Proclin300 is added to obtain highly pure sheep anti-human Lp(a) polyclonal antibodies. Purity test as follows Figure 2 .

[0041] Example 8 Lp(a) polyclonal antibody validation

[0042] Prepare an Lp(a) immunoturbidimetric kit to test the calibrator value, wherein the reagent R1 includes a buffer 1 (e.g., phosphate, citrate), a surfactant (e.g., Triton X100), a stabilizer (e.g., NaCl), a chelating agent (e.g., EDTA), a sensitizer (e.g., PEG6000), and a preservative (e.g., NaN3)

[0043] Reagent R2: Dilute the above-mentioned sheep anti-human Lp(a) polyclonal antibody and the commercial sheep anti-human Lp(a) polyclonal antibody purchased from manufacturer A with PBS buffer 1 (pH 7-8) to the same concentration of 10-20 mg / mL; dilute latex microspheres (carboxylated polystyrene latex, purchased from Ninebot, particle size 120 nm) with purified water to 5% (solid content), then add 5-10% (EDC / latex microsphere mass ratio) of EDC, stir and react at 25-30°C for 20-30 min, then add the diluted antibody (1 mL of latex microspheres corresponds to 10-16 mg of antibody), stir and react at 25-30°C for 90-150 min, add the stop reaction solution to stop the reaction, centrifuge to remove the supernatant, add a buffer (e.g., phosphate, citrate), a surfactant (e.g., Triton X100), a stabilizer (e.g., NaCl), a chelating agent (e.g., EDTA), a protective agent (e.g., BSA) and a preservative (e.g., NaN3) are added to the buffer 2, and the R2 reagent is obtained after ultrasonic treatment for 120-180S.

[0044] Calibrators: A buffer consisting of buffer 3 (e.g. phosphate, citrate), a stabilizer (e.g. NaCl), a chelating agent (e.g. EDTA) and a preservative (e.g. NaN3) was added to different volumes of natural human Lp(a) protein to prepare a set of calibrators with final concentrations of 1000, 500, 250, 125, 62.5 and 0 mg / L (purified water was used as a control). In addition, a high-value test product of 625 mg / L was prepared.

[0045] Kit performance test: Control kit, the antibody used in reagent 2 is the commercial sheep anti-human polyclonal antibody of manufacturer A, and the other parameters are the same, and the test parameters are: main wavelength 600nm, sub-wavelength none, R1:R2:S=200:50:3. Test instrument: Hitachi 3500.

[0046] (1) Linear range verification: Use the kit of the present invention and the control kit to measure each calibrator and a high-value test product. Use the concentration of the calibrator as the independent variable and the absorbance as the dependent variable to obtain a working curve. Figure 3The results show that the detection range of the kit of the present invention is 0-1000 mg / mL, and the linear correlation coefficient can reach 0.9999. It can be seen that the kit of the present invention has a wider linear range, which is more conducive to the direct detection of high-concentration pathological samples without the need for sample dilution, and the operation is simplified.

[0047] (2) Antibody specificity (analytical sensitivity) verification: The test kit of the present invention and the control kit were used to repeatedly measure the sample of (227.0±30) mg / L for 3 times, and the absorbance A generated by the reagent under the specified parameters was recorded, and the absorbance difference (ΔA) of 3.0 mg / L was converted according to formula (1), as shown in Table 1. It can be seen from the data in the table that when testing the sample of 227.0 mg / L, the kit of the present invention has a higher analytical sensitivity, that is, a better specificity.

[0048]

[0049] Table 1

[0050]

[0051] (3) Detection limit verification: The blank sample was repeatedly measured 20 times using the kit of the present invention and the control kit, and the mean value (M) and standard deviation (SD) were calculated. The detection limit = M-2SD, see Table 2. As can be seen from the data in the table, the kit of the present invention has a lower detection limit, that is, a higher sensitivity.

[0052] Table 2

[0053] Kit of the present invention Control Kit Test value 1 0.68 0.27 Test value 2 0.2 -5.7 Test value 3 0.35 1.86 Test value 4 0.22 -0.91 Test value 5 0.2 1.17 Test value 6 0.13 0.81 Test value 7 0.07 -1.56 Test value 8 0.43 -0.21 Test value 9 0.25 3.8 Test value 10 0.83 0.50 Test value 11 0.01 0.83 Test value 12 0.72 1.89 Test value 13 0.35 0.23 Test value 14 0.7 1.38 Test value 15 0.05 1.62 Test value 16 0.17 2.07 Test value 17 0.16 2.97 Test value 18 0.26 1.29 Test value 19 0.8 1.14 Test value 20 0.86 1.32 Average value X 0.372 0.739 Standard Deviation 0.285 1.944 Detection limit -0.197 -3.149

[0054] The above disclosure is only a preferred embodiment of the method for preparing recombinant human lipoprotein a and its polyclonal antibody of the present invention, and it is certainly not used to limit the scope of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for preparing recombinant human lipoprotein a and its polyclonal antibody, characterized in that: The following steps are involved: Using pET28a(+) as the expression vector, the Lp(a) gene with 6 His tags, Trx tags and PPase cleavage sites fused to the N-terminus under the control of T7 promoter was introduced into Escherichia coli for induction, and then the inclusion bodies were denatured and renatured and purified by NiFocurose 6FF column to obtain Lp(a) fusion protein with a purity of ≥90%; The Lp(a) fusion protein was cleaved and purified by Ni Focurose 6FF column to obtain Lp(a) protein without tag with a purity of ≥95%; After goats were immunized with Lp(a) protein with a purity of ≥95%, serum was collected after each immunization, and qualified serum was screened by antiserum two-way agar diffusion experiment, and purified using Protein A to obtain Lp(a) polyclonal antibodies.

2. The method for preparing a recombinant human lipoprotein a and a polyclonal antibody thereof according to claim 1, characterized in that: The Escherichia coli is Ecoli BL21 (DE3).

3. The method for preparing a recombinant human lipoprotein a and a polyclonal antibody thereof according to claim 1, characterized in that: The E. coli fermentation conditions are as follows: inoculating the seed liquid into 1000 ml fermentation medium containing 100 ug / mL kan at a 5% inoculation rate, expanding the culture at 37° C. and 220 rpm for about 1-2 hours, adding IPTG when OD600 rises to 0.8 to make the final concentration of IPTG in the fermentation liquid 0.5 mM / L, and continuing to culture in a shaker at 18° C. and 220 rpm for 12-14 hours.

4. The method for preparing a recombinant human lipoprotein a and a polyclonal antibody thereof according to claim 3, characterized in that: The fermentation medium contains 15 g / L of tryptone, 25 g / L of yeast powder, 0.6 g / L of MgSO4, 23 g / L of Na2HPO4, 5 g / L of NaH2PO4 and 15 g / L of glucose, and the pH value is 7.2.

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