CN113046324A

CN113046324A - Pepsinogen II recombinant protein and monoclonal antibody thereof, preparation method and application

Info

Publication number: CN113046324A
Application number: CN202110390444.0A
Authority: CN
Inventors: 黎榕萍
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-04-12
Filing date: 2021-04-12
Publication date: 2021-06-29

Abstract

The invention discloses a CHO-K1 cell strain, wherein the CHO-K1 cell strain contains a gene capable of efficiently secreting and expressing PG II recombinant protein. The invention also discloses a method for preparing the CHO-K1 cell strain, which comprises the following steps: 1) cloning a gene sequence shown in SEQ ID NO.1 into a eukaryotic expression vector to obtain a recombinant plasmid containing a PG II recombinant protein coding gene; 2) transfecting the recombinant plasmid into CHO-K1 cells to obtain a CHO-K1 cell strain; 3) culturing, screening and domesticating the CHO-K1 cell strain in the step 2) to obtain a cell strain which can efficiently secrete and express PG II recombinant protein. The invention also discloses an anti-PG II monoclonal antibody, wherein the combined epitope of the monoclonal antibody 1 is positioned at aa78-aa90 of pepsinogen II; the epitope bound by the monoclonal antibody 2 is positioned at aa280-aa291 of pepsinogen II. The protein of the invention has high expression quantity, good quality and low cost; the monoclonal antibody can be used for detecting PG II protein in a matched mode, and is good in specificity and high in sensitivity.

Description

Pepsinogen II recombinant protein and monoclonal antibody thereof, preparation method and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to pepsinogen II recombinant protein, a monoclonal antibody thereof and a preparation method thereof.

Background

Pepsinogen (PG), an endoprotease with digestion function, is a single-chain polypeptide consisting of 375 amino acids, belonging to the aspartic protein family. Human pepsinogen can be divided into seven isozymogens pgl-PG7 from fast to slow according to the mobility under the agar gel electrophoresis, wherein pgl-PG5 has common immunogenicity and is also called PG I; the mobility is slower for PG6 to PG7, also known as PG II. In vivo, PG I and PG II have different cell sources and tissue distributions. Research shows that the reduction of PG I level is a reliable sign for the occurrence and development of gastropathy. The low-level serum PG is also regarded as a sign for the occurrence and development of stomach diseases abroad, and epidemiological research shows that the serum PG has a wider atrophy range of gastric mucosa and is obviously reduced especially when the stomach body is affected. Therefore, the detection of PG I and PG II is of great clinical significance.

At present, there are many diagnostic reagents for PG II detection on the market, and methodologies mainly focus on immunological diagnosis, specifically colloidal gold, chemiluminescence, and biochemical turbidimetry. The premise behind the use of these methods is the PG II protein and the adapted antibody. At present, the PG II protein is mainly obtained in 2 modes, namely, the PG II protein is purified from human serum, and the PG II protein is expressed in vitro by a gene recombination method. The first method is relatively expensive because it is laborious and time consuming, and the second method considers a suitable expression system to make the expressed protein closer to the PG II protein in human body. Aiming at the detection of the PG II protein, the specificity and the sensitivity of the monoclonal antibody are more and better, so that the screening of the proper monoclonal antibody plays an important role in the diagnosis and the detection of the PG II protein.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a PG II recombinant egg, a related monoclonal antibody, a preparation method and application.

Therefore, the invention provides a CHO-K1 cell line, wherein the CHO-K1 cell line contains a gene capable of efficiently secreting and expressing pepsinogen II recombinant protein.

Preferably, the pepsinogen II recombinant protein gene is a nucleotide sequence after codon optimization.

Preferably, the codon-optimized nucleotide sequence of the invention is shown in SEQ ID NO. 1.

In another aspect, the invention also provides a method for preparing the CHO-K1 cell strain, which comprises the following steps: 1) cloning a gene sequence shown in SEQ ID NO.1 into a eukaryotic expression vector to obtain a recombinant plasmid containing a pepsinogen II recombinant protein coding gene; 2) transfecting the recombinant plasmid into CHO-K1 cells to obtain a CHO-K1 cell strain; 3) culturing, screening and domesticating the CHO-K1 cell strain in the step 2) to obtain the cell strain which can efficiently secrete and express pepsinogen II recombinant protein.

Preferably, the eukaryotic expression vector of the invention is pee 12.4.

In still another aspect, the present invention provides an anti-pepsinogen II monoclonal antibody, wherein the anti-pepsinogen II monoclonal antibody is prepared by using pepsinogen II recombinant protein expressed by the CHO-K1 cell strain of claim 1, and the combined epitope of the monoclonal antibody 1 is positioned at aa78-aa90 of pepsinogen II; the amino acid sequence of aa78-aa90 is SerIleGlyThrProGlnAsPheLeuValLeuPhe; the epitope combined by the monoclonal antibody 2 is positioned at aa280-aa291 of pepsinogen II; the amino acid sequence of aa280-aa291 is SerLeuThrValProGlnGlnTyrMetSerAla.

Preferably, the monoclonal antibodies of the invention are all of the IgG1 subtype.

In still another aspect, the invention also provides an application of the CHO-K1 cell strain in large-scale preparation of pepsinogen II recombinant protein.

In still another aspect, the invention also provides the use of a monoclonal antibody in the preparation of a diagnostic reagent for pepsinogen II.

The invention constructs and screens a CHO-K1 cell strain which can stably and efficiently secrete and express pepsinogen II recombinant protein in a suspended manner, the cell strain expresses PG II protein with high yield (the yield is as high as 0.5g/L or more), is easy to purify (PG II protein is secreted and expressed, the purity of target protein can reach 90% or more only by passing a tweezer column, and the step of removing endotoxin is not needed), and is easy for large-scale production (the fermentation scale can reach 100L or more in industrial production). Therefore, the invention not only provides a method for large-scale industrial production of the pepsinogen II recombinant protein, but also greatly reduces the cost for preparing the pepsinogen II recombinant protein at present. In addition, the PG II protein prepared by the method is a eukaryotic cell strain which is closer to the PG II protein in a human body compared with baculovirus expression, and the cell strain has high controllability, easy quality control and simple quantification during culture, so the PG II protein prepared by the method also has the following advantages: large-scale industrial production can be realized, the supply is sufficient, and the quality control is easy; the batch to batch stability; the biosafety control in the production is easy (serum-free culture medium is used in the fermentation culture, and the risk of toxin dispersion does not exist).

The monoclonal antibody of anti-PG II protein is prepared by using the PG II protein prepared by the invention, 2 strains of suitable monoclonal antibodies are screened together, and the epitope recognized by the monoclonal antibodies is researched, wherein the epitope recognized by the 2 strains of monoclonal antibodies is far in position and is suitable for being matched and used for detecting the PG II protein. And experiments prove that (see example 3), a diagnosis system established by using the PG II protein and the two monoclonal antibodies has the characteristics of high sensitivity and good specificity, and has good conformity with imported reagents.

Drawings

FIG. 1 shows SDS-PAGE patterns of PG II proteins, wherein M is Marker, PG II proteins 7-10 are expressed by 120 strains, and PG II

proteins

9 and 10 are expressed by 50 strains.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only some of the embodiments of the invention, and not all of them.

The chemical reagents related to the invention are all made in China.

EXAMPLE 1 preparation of pepsinogen II

Cloning the nucleotide sequence (the specific sequence is shown as SEQ ID NO. 1) of the pepsinogen II after codon optimization into a eukaryotic expression vector (for example). The nucleotide sequence (the specific sequence is shown as SEQ ID NO. 1) of the codon-optimized pepsinogen II is taken as a template, and EcoRI and HindIII double enzyme cutting sites are respectively used for connection to construct pEE12.4-PG II-6 His expression plasmid. The identified positive recombinant plasmid is sent to Huada biology company for sequence determination, the determined nucleotide sequence and the coded amino acid sequence are analyzed and compared by software, and the correctness of the reading frame is checked.

CHO-K1 cells were transfected with the correctly identified pEE12.4-PG II-6 His expression plasmid. Pressure selection was started 24h after transfection: six well plate cells were removed from the 37 ℃ incubator, the supernatant medium was discarded, 2mL of DMEM/F12 (containing 10% serum + 25. mu.M MSX) was added, the pressure was increased for 7 days, the cells were observed in the middle, and the dead cells were replaced with more cells. Monoclonal screening was performed by pressure screening until the negative control cells were essentially dead (about 7 days): the six well plate was removed, the medium was discarded, PBS was washed once, then 300. mu.L of 0.25% trypsin-EDTA was added, the digestion was carried out at room temperature for about 2min, 2mL of DMEM/F12 (containing 10% serum + 25. mu.M MSX) was added to stop the digestion reaction, and the cells were blown off by pipette. The digested cells were transferred to a 15mL centrifuge tube and centrifuged at room temperature for 5min at 200 g. Cells were resuspended in DMEM/F12 (containing 10% serum + 25. mu.M MSX) and counted. Plate paving: diluting the cells to 5/mL, adding 200. mu.L of the mixed cells to a 96-well plate, standing at 37 ℃ with 5% CO₂And incubating for 4-6h in the cell incubator. Wells of individual cells were recorded. When the wells of the individual cells in the 96-well plate were grown up, the medium was discarded, PBS was washed once, 100mL of 0.25% trypsin-EDTA was added, digestion was carried out at room temperature for about 2min, 2mL of DMEM/F12 (containing 10% serum + 25. mu.M MSX) was added to stop the digestion reaction, and the cells were blown off by pipette. And transferring the cell sap to a 12-pore plate, taking the supernatant when the 12-pore plate is full, detecting whether the clone is positive by ELISA, and continuously performing expanded culture and freezing storage on the high-efficiency expressed positive clone. After screening, 2 cell lines, 120 and 50 cell lines were harvested.

Screening the monoclonal cellsDomesticating the strain into suspension culture: the cells were removed from the 37 ℃ incubator, the supernatant medium was discarded, the cells were washed once with pre-warmed 8mL PBS, and the PBS was discarded. Adding 1-2mL of 0.25% trypsin-EDTA into each 10cm cell culture dish, digesting at room temperature for about 2min, observing the cells under a microscope to shrink and become round, and displaying the cells as single cells. Digestion was stopped by adding 4mL of DMEM/F12 (10% serum, 25. mu.M MSX) and the cells were blown off with a pipette gun. The digested cells were transferred to a 15mL centrifuge tube and centrifuged at room temperature for 5min at 200 g. Cells were suspended in 100% DMEM/F12 (containing 10% serum, 25. mu.M MSX) and counted. Dilute cells to 5 × 10⁵cells/mL were inoculated in 30mL culture medium in a 125mL shake flask. The cell culture flask was placed at 37 ℃ with 5% CO₂Incubate overnight at 120r/min on an orbital shaker in a cell incubator. Wiping the biological safety cabinet table top with 75% alcohol for sterilization, and irradiating with ultraviolet for 30 min. Cell density and viability were counted every 24 h. And performing second-generation culture when the cell survival rate reaches 94-97% after the first-generation cell culture is performed once. Preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; 100% DMEM/F12 (containing 10% serum, 25. mu.M MSX), EX-CELL 302 was preheated to 37 ℃ in a CO2 CELL incubator. The cells were removed from the 37 ℃ incubator, transferred to a 50mL centrifuge tube, and centrifuged at 200g for 5min at room temperature. DMEM/F12 (containing 10% serum, 25. mu.M MSX) and EX-CELL 302 were mixed well at a ratio of 1:1.5, the CELLs were resuspended and counted. Dilute cells to 5 × 10⁵cells/mL were inoculated in 30mL culture medium in a 125mL shake flask. The cell culture flask was placed at 37 ℃ with 5% CO₂Incubate overnight at 120r/min on an orbital shaker in a cell incubator. Wiping the biological safety cabinet table top with 75% alcohol for sterilization, and irradiating with ultraviolet for 30 min. Cell density and viability were counted every 24 h. The survival rate of the cells obtained after the second generation culture is twice is more than 95 percent; the cell survival rate obtained after three times of culture of the third to the sixth generation is more than 95 percent. After 7 weeks, the cells were seeded for 3 days and propagated for three generations with a density of 1X 10⁶Individual cells/mL with a cell viability of 95%, which cells are considered to have been adapted to suspension culture. The inoculation density is reduced to 3 x 10⁵one/mL. After acclimation, 120 strains and 50 strains meet the requirements, which shows that the acclimation of 120 strains and 50 strains is successful.

Performing shake flask fermentation culture on 120 and 50 CHO-K1 cells, harvesting cell culture supernatant after 12 days, and performing affinity purification and secretion expression on PG II-6 His protein by using a nickel column (GE medical treatment); the concentration was determined with BCA kit (Byunnan), and the purity was determined with SDS-PAGE. The expression yield can reach 500mg/L or more, and the purity of SDS-PAGE (as shown in figure 1, the molecular weight of the expressed PG II protein is about 53kDa, the theoretical molecular weight of the PG II protein is 42.6kDa, and the difference between the two is caused by the PG II protein after translation modification in CHO-K1 cells, and both are glycosylation results) is more than 90%. The protein after determination is frozen and stored at-80 ℃ for standby. The results show that 120 and 50 CHO-K1 cell strains subjected to monoclonality and suspension are suitable for large-scale fermentation production, and have the advantages of low cost (high yield), good quality (high purity, eukaryotic expression system with post-translational modification function, closest approach to human source) and good batch-to-batch stability (the same cell strain is fermented and purified to ensure that the expressed proteins are basically consistent). Wherein the amino acid sequence of the expressed pepsinogen II is shown as SEQ ID NO. 2.

EXAMPLE 2 preparation of monoclonal antibody against pepsinogen II

The 8-week-old BALB/c mice are immunized by the pepsinogen II recombinant protein prepared in the example 1, when the mice are immunized for the first time, the pepsinogen II recombinant protein and Freund's complete adjuvant are emulsified in equal volume, and the mice are inoculated with 100 mu g of protein per mouse; after 7 days, the pepsinogen II recombinant protein and Freund's incomplete adjuvant are emulsified in equal volume, and the mice are immunized by the second abdominal cavity inoculation way, wherein each mouse contains 100 mu g of protein; 7 days later, the third mouse abdominal cavity approach directly immunizes 100 mu g/mouse pepsinogen II recombinant protein; on the 3 rd day after immunization, fusion of mouse spleen cells and mouse myeloma cells SP2/0 is carried out, and HAT selective culture medium is cultured; after 10 days, taking pepsinogen II recombinant protein as a coating antigen, detecting cell supernatant by indirect ELISA, screening positive hybridoma cells, and screening 47 cell strains and 118 cell strains.

Injecting 0.5mL of pristane into the abdominal cavity of 8-10-week-old Balb/c mice, and injecting 1 × 10 hybridoma cells (47 strains and 118 strains are respectively cultured and injected) into each mouse 7-10 days later⁶～2×10⁶7-10 days later, extracting ascites from the mice,centrifuging at 1200r/min for 10 minutes at the temperature of 2-8 ℃, and collecting supernatant. The monoclonal antibodies were purified using a ProteinG affinity column and the antibodies were split into 0.5 mL/tube and stored at-20 ℃ until needed.

Next, we entrusted Nanjing Kinshire company to perform typing detection on the monoclonal antibody and detect the recognized epitope, and the results show that the two monoclonal antibodies against pepsinogen II of the invention are IgG1 subtype, wherein the epitope bound by 47 strains is located at positions aa78-aa90 of pepsinogen II (the amino acid sequence is specifically SerIleGlyThrProProGlnSnPheuValLeuPhe); the epitope bound by the 118 strain is positioned at aa280-aa291 of pepsinogen II (the amino acid sequence is specifically SerLeuThrValProGlnGlnTyrMetSerAlA).

EXAMPLE 3 application of pepsinogen II and anti-pepsinogen II monoclonal antibodies

The PG II protein prepared by the invention is used as a calibrator, the two monoclonal antibodies prepared by the invention are used as detection antibodies, and a set of diagnostic reagents for the PG II protein is established, wherein the specific reagent preparation method is shown in CN 109307765A. The results of performance comparisons using the reagent of the invention with yapei's kit are shown in the following table: the diagnostic reagent prepared by the PG II protein and the monoclonal antibody can be used for detecting the PG II protein, and has good detection result (good specificity and high sensitivity), and the coincidence rate with an imported reagent is up to 96.8 percent.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Sequence listing

<110> Ficus benjamina

<120> pepsinogen II recombinant protein, monoclonal antibody thereof, preparation method and application

<160>2

<170>SIPOSequenceListing 1.0

<210>2

<211>388

<212>PRT

<213> pepsinogen II amino acid sequence ()

<400>2

Met Lys Trp Met Val Val Val Leu Val Cys Leu Gln Leu Leu Glu Ala

1 5 10 15

Ala Val Val Lys Val Pro Leu Lys Lys Phe Lys Ser Ile Arg Glu Thr

20 25 30

Met Lys Glu Lys Gly Leu Leu Gly Glu Phe Leu Arg Thr His Lys Tyr

35 40 45

Asp Pro Ala Trp Lys Tyr Arg Phe Gly Asp Leu Ser Val Thr Tyr Glu

50 55 60

Pro Met Ala Tyr Met Asp Ala Ala Tyr Phe Gly Glu Ile Ser Ile Gly

65 70 75 80

Thr Pro Pro Gln Asn Phe Leu Val Leu Phe Asp Thr Gly Ser Ser Asn

85 90 95

Leu Trp Val Pro Ser Val Tyr Cys Gln Ser Gln Ala Cys Thr Ser His

100 105 110

Ser Arg Phe Asn Pro Ser Glu Ser Ser Thr Tyr Ser Thr Asn Gly Gln

115 120 125

Thr Phe Ser Leu Gln Tyr Gly Ser Gly Ser Leu Thr Gly Phe Phe Gly

130 135 140

Tyr Asp Thr Leu Thr Val Gln Ser Ile Gln Val Pro Asn Gln Glu Phe

145 150 155 160

Gly Leu Ser Glu Asn Glu Pro Gly Thr Asn Phe Val Tyr Ala Gln Phe

165 170 175

Asp Gly Ile Met Gly Leu Ala Tyr Pro Ala Leu Ser Val Asp Glu Ala

180 185 190

Thr Thr Ala Met Gln Gly Met Val Gln Glu Gly Ala Leu Thr Ser Pro

195 200 205

Val Phe Ser Val Tyr Leu Ser Asn Gln Gln Gly Ser Ser Gly Gly Ala

210 215 220

Val Val Phe Gly Gly Val Asp Ser Ser Leu Tyr Thr Gly Gln Ile Tyr

225 230 235 240

Trp Ala Pro Val Thr Gln Glu Leu Tyr Trp Gln Ile Gly Ile Glu Glu

245 250 255

Phe Leu Ile Gly Gly Gln Ala Ser Gly Trp Cys Ser Glu Gly Cys Gln

260 265 270

Ala Ile Val Asp Thr Gly Thr Ser Leu Leu Thr Val Pro Gln Gln Tyr

275 280 285

Met Ser Ala Leu Leu Gln Ala Thr Gly Ala Gln Glu Asp Glu Tyr Gly

290 295 300

Gln Phe Leu Val Asn Cys Asn Ser Ile Gln Asn Leu Pro Ser Leu Thr

305 310 315 320

Phe Ile Ile Asn Gly Val Glu Phe Pro Leu Pro Pro Ser Ser Tyr Ile

325 330 335

Leu Ser Asn Asn Gly Tyr Cys Thr Val Gly Val Glu Pro Thr Tyr Leu

340 345 350

Ser Ser Gln Asn Gly Gln Pro Leu Trp Ile Leu Gly Asp Val Phe Leu

355 360 365

Arg Ser Tyr Tyr Ser Val Tyr Asp Leu Gly Asn Asn Arg Val Gly Phe

370 375 380

Ala Thr Ala Ala

385

<210>3

<211>1164

<212>DNA

<213> Codon-optimized pepsinogen II nucleotide sequence (Codon optimized pepsinogen)

<400>3

atgaaatgga tggtggtggt gctcgtatgc ctgcaactgc tcgaagccgc ggtggtaaag 60

gtgcccctga aaaagttcaa atccatccgg gagactatga aggagaaagg cctcctgggg 120

gaatttctgc gaacccacaa gtacgacccc gcatggaagt acagatttgg ggatctctct 180

gtgacctacg agcccatggc atacatggat gccgcctact tcggcgaaat ctccattggg 240

acaccccccc agaatttttt ggtcttgttc gatactgggt cttcaaattt gtgggtgccg 300

tctgtgtact gtcagtccca ggcctgtacg tcccacagta ggtttaatcc tagtgaatct 360

tctacctatt ctaccaatgg gcagaccttc agtctccagt atgggtctgg aagcctcaca 420

ggattctttg gatacgatac attgacagtg cagagcatcc aggtccccaa ccaggaattt 480

ggcctttctg agaatgaacc aggcacaaac ttcgtctacg cccagtttga cgggattatg 540

ggcttggcct atccagccct gtcagtcgat gaagccacaa ccgcaatgca ggggatggta 600

caggaaggcg cgctcacctc cccagtcttc agcgtttacc tgtcaaacca acagggatcc 660

tccggtggtg cagttgtttt tggcggagta gattcaagcc tgtatacagg tcagatttac 720

tgggcccccg tgacgcagga gctgtactgg cagattggca ttgaggagtt cctcattggg 780

ggccaggcta gcggctggtg cagcgaaggc tgtcaggcca tagtagacac cgggacctca 840

ctgttgaccg ttcctcagca atacatgtcc gccctgctgc aagccactgg tgcccaggag 900

gacgagtatg ggcaattcct ggtcaactgc aatagcatcc agaatctgcc cagcctcact 960

ttcatcatca acggggtgga gtttccactc ccccccagtt cttatatact cagtaataat 1020

ggatattgca ccgtcggcgt ggaaccaacc tatctgtctt ctcaaaacgg gcaaccactc 1080

tggatcctgg gtgatgtgtt ccttagatct tattacagcg tgtatgatct ggggaacaac 1140

agagtggggt ttgctaccgc ggcc 1164

Claims

1. A CHO-K1 cell strain, characterized in that, the CHO-K1 cell strain contains a gene which can efficiently secrete and express pepsinogen II recombinant protein.

2. The CHO-K1 cell line of claim 1, wherein the pepsinogen II recombinant protein gene is a codon optimized nucleotide sequence.

3. The CHO-K1 cell line according to claim 2, wherein the codon-optimized nucleotide sequence is shown in SEQ ID No. 1.

4. A method for preparing the CHO-K1 cell line according to any one of claims 1 to 3, wherein the method comprises the following steps:

1) cloning a gene sequence shown in SEQ ID NO.1 into a eukaryotic expression vector to obtain a recombinant plasmid containing a pepsinogen II recombinant protein coding gene;

2) transfecting the recombinant plasmid into CHO-K1 cells to obtain a CHO-K1 cell strain;

3) culturing, screening and domesticating the CHO-K1 cell strain in the step 2) to obtain the cell strain which can efficiently secrete and express pepsinogen II recombinant protein.

5. The method of claim 4, wherein the eukaryotic expression vector is pEE12.4.

6. An anti-pepsinogen II monoclonal antibody, wherein the anti-pepsinogen II monoclonal antibody is prepared by using pepsinogen II recombinant protein expressed by the CHO-K1 cell strain of claim 1, and the combined epitope of the monoclonal antibody 1 is positioned at aa78-aa90 of pepsinogen II; the amino acid sequence of aa78-aa90 is SerIleGlyThrProGlnAsPheLeuValLeuP he; the epitope combined by the monoclonal antibody 2 is positioned at aa280-aa291 of pepsinogen II; the amino acid sequence of aa280-aa291 is SerLeuThrValProGlnGlnTyrMetSerAla.

7. The monoclonal antibody of claim 6, wherein each of said monoclonal antibodies is of the IgG1 subtype.

8. Use of the CHO-K1 cell line according to any one of claims 1 to 3 for large-scale production of pepsinogen II recombinant protein.

9. Use of a monoclonal antibody according to any one of claims 6 to 7 in the preparation of a diagnostic reagent for pepsinogen II.