CN115850473B

CN115850473B - IL-8 antibodies and uses thereof

Info

Publication number: CN115850473B
Application number: CN202211499692.XA
Authority: CN
Inventors: 陈小平; 龙凤英; 徐义; 杜胤骁
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2022-11-28
Filing date: 2022-11-28
Publication date: 2023-12-08
Anticipated expiration: 2042-11-28
Also published as: CN115850473A

Abstract

The invention discloses an IL-8 antibody and application thereof. The invention takes human IL-8 as antigen, and rabbits as immune subjects, through subcutaneous injection, after a plurality of rounds of immunization, spleen of the rabbits is extracted, mononuclear cells are separated, total RNA is extracted, reverse transcription is carried out to cDNA, and specific primers are used for amplification to obtain all heavy chain variable regions and light chain variable regions, so as to construct phage antibody libraries. Monoclonal antibodies that bind to IL-8 were screened by phage antibody libraries. The anti-IL-8 monoclonal antibodies of the invention specifically bind to IL-8 and are useful for diagnosing or treating diseases associated with abnormal IL-8 expression.

Description

IL-8 antibodies and uses thereof

Technical Field

The invention belongs to the technical field of screening, preparation and application of monoclonal antibodies, and particularly relates to an anti-IL-8 monoclonal antibody and application thereof.

Background

Interleukin-8 (IL-8), also known as CXCL8, belongs to the CXC subtype and is the earliest chemotactic cytokine discovered. IL-8 is divided into two subgroups, alpha and beta, the IL-8 initial translation product is 99 amino acids, cut into 72 amino acids in mononuclear and macrophage cells, and the molecular weight is 8kDa active substance. IL-8 is secreted primarily by monocytes and macrophages and functions to recruit and activate neutrophils, contributing to the inflammatory response and cell killing. In addition, tumor cells, fibroblasts, endothelial cells and the like affect proliferation, invasion, metastasis of tumor cells and formation of tumor blood vessels through autocrine or paracrine under the action of pro-inflammatory factors such as IL-1 beta, tumor necrosis factor alpha, lipopolysaccharide and the like.

IL-8 functions by binding to the IL-8 receptor, and IL-8 has 2 receptors, CXCR1 (IL-8 RA) and CXCR2 (IL-8 RB), respectively, which are 77% homologous, all belonging to the G-protein coupled receptor. Because of the structural differences at the N-terminus, 2 receptors have different affinities for IL-8, CXCR1 having a higher affinity for IL-8. In normal tissue cells, CXCR1 first binds granulocyte chemotactic protein 2 (GCP-2) to form a complex and then binds IL-8 to function; CXCR2 has a lower affinity for IL-8 and CXCR2 can bind to a variety of ligands such as CXCL1, 2, 3, 5, 7 in addition to IL-8. IL-8 has obvious chemotaxis and function activation effects on neutrophils, can induce chemotaxis of CD4+ and CD8+ T cells, and is involved in the development of various diseases and tissue injury, such as rheumatoid arthritis, psoriasis, ischemia reperfusion syndrome (including myocardial infarction and multi-organ failure), glomerulonephritis and other various infectious diseases; IL-8 binding to the receptor can promote tumor development by activating multiple downstream signaling pathways such as PI3K/Akt, PLC/PKC, MAPK, and the like. Thus screening for IL-8 antagonistic monoclonal antibodies is effective in the treatment of IL-8 mediated diseases.

The IL-8 is used as immunogen to immunize rabbit, and the anti-IL-8 phage antibody library is constructed, so that the anti-IL-8 antibody can be effectively screened, and the anti-IL-8 antibody can be produced in large scale by genetic engineering and protein expression technology, and can be used for diagnosing and treating diseases related to IL-8 abnormality.

The invention comprises the following steps:

the main object of the present invention is to provide specific monoclonal antibodies against IL-8 and uses thereof. Spleen is obtained from immunized rabbits, an anti-IL-8 phage antibody library is constructed, and antibodies aiming at IL-8 are rapidly screened, so that the antibodies can be used for diagnosing and treating diseases caused by abnormal IL-8 expression.

The IL-8 antibodies screened according to the invention comprise: YX20

YX20 heavy chain CDR sequence:

CDRH1 amino acid sequence GFSLNNYA, shown in SEQ NO.1;

CDRH2 amino acid sequence VGSDDIP, see SEQ NO.2

The amino acid sequence ASGYVGDDRYNI of CDRH3 is shown in SEQ NO.3;

YX20 light chain CDR sequence:

CDRL1 amino acid sequence PSVYNNY, see SEQ NO.4;

CDRL2 amino acid sequence AAS, see SEQ NO.5;

the amino acid sequence AGAYSNDSDDG of CDRL3 is shown in SEQ NO.6.

YX20 heavy chain variable region amino acid sequence:

GSTGDQEQLVESGGRLVTPGTPLTLTCTVSGFSLNNYAMSWVRQAPGKGLEYIGVVGSDDIPFYA SWAKGRFAISKTSTTVDLKITSPTTEDTATYFCASGYVGDDRYNIWGPGTLVTISS, SEQ NO.7;

YX20 light chain variable region amino acid sequence:

GSTGDAAVVLTQTPSPVSAAVGGTVSISCQSSPSVYNNNYLSWYQQKPGQPPKLLIYAASTLASG VPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGAYSNDSDDGFGGGTKLEIK, SEQ NO.8.

Such antibody molecules include, but are not limited to: immunoglobulin, fab, (Fab') 2 or single chain antibody.

The IL-8 antibody is used for preparing a preparation for diagnosing the abnormal expression of IL-8.

The IL-8 antibody is used for preparing and treating diseases related to IL-8 expression abnormality.

Diseases associated with aberrant IL-8 expression include tumors.

The invention provides prokaryotic expression of IL-8, injecting rabbit subcutaneously according to 1 mg/dose to obtain IL-8, immunizing for 4 rounds at intervals of 2 weeks, killing rabbit after the end of immunization, picking spleen, grinding spleen into single cell suspension, separating to obtain mononuclear cells, obtaining mononuclear cells and extracting total RNA, synthesizing cDNA, using heavy chain variable region and light chain variable region merger primer, amplifying heavy chain variable region and light chain variable region, randomly combining obtained heavy chain variable region and light chain variable region, recombining with phage vector, converting XL1-Blue by recombinant product, adding auxiliary phage VCSM13 to obtain titer of about 10 ¹³ A large phage antibody library. The antigen IL-8 is used for screening an anti-IL-8 antibody, and the heavy chain variable region and the light chain variable region which can be combined with the IL-8 are obtained through sequencing, wherein the heavy chain variable region is combined with an Fc fragment of human IgG1, and the heavy chain and the light chain of the antibody are simultaneously expressed in mammalian cells to express the humanized and modified anti-IL-8 antibody. Through in vitro experiments, the anti-IL-8 monoclonal antibody screened by the invention can be specifically combined with IL-8, and can be used for detecting or treating diseases caused by abnormal IL-8 expression.

Description of the drawings:

FIG. 1IL-8 gene cloning;

FIG. 2 detection by SDS-PAGE of purified IL-8;

FIG. 3 heavy chain variable region gene cloning of anti-IL 8 antibodies;

FIG. 4 cloning of anti-IL 8 antibody light chain variable region genes;

FIG. 5 heavy and light chain variable region gene splice of anti-IL 8 antibody;

FIG. 6 identification of anti-IL 8 phage positive clones;

FIG. 7ELISA identifies a round 5 screening phage library;

FIG. 8 PCR identification of antibody heavy and light chains with pcDNA3.4 ligation;

FIG. 9 SDS-PAGE electrophoresis of purified antibody YX 20;

FIG. 10 inhibition of neutrophil chemotaxis by anti-IL 8 antibodies;

FIG. 11 anti-IL 8 antibody inhibits HCT116 growth in vivo.

The specific embodiment is as follows:

the invention is further described below in connection with specific examples without limiting the invention.

The IL-8 protein used in the examples of the present invention was prepared by the laboratory itself.

The phage antibody library construction method of the invention is referred to: antonny S.Dimitrov (ed.), generation and Selection of Rabbit Antibody Libraries by Phage Display,2009, therapeutic μtic Antibodies: methods and Protocols, vol.525, 101-128.

Example 1

1.IL-8 protein production

The human monocyte cDNA is used as template, the primers IL-8F and IL-8R are used to clone IL-8 fragment, the fragment is led into pGEX-4T-1 vector (vast, P0001), pGEX-4T-1-IL-8 which is successfully constructed is obtained through screening, expressed under IPTG induction condition, and IL-8 protein is obtained through purification.

The method specifically comprises the following steps:

1. cloning of IL-8 fragments

Dilution of monocyte cDNA 10-fold

(1) The system was formulated as follows in table 1:

TABLE 1

cDNA	2μl
		IL-8F	4μl
IL-8R	4μl
		ddH ₂ O	40μl
PrimerSTAR (from TAKARA)	50μl
		Total	100μl

Note that: cloning of IL8 Gene primer

IL8-F: GTTCCGCGTGGATCCCCGGAAGAAGGTGCAGTTTTGCCAAG, see SEQ NO.9,

IL8-R: TCGAGTCGACCCGGGAATTTCATGAATTCTCAGCCCTCTTCA, SEQ NO.10;

(2) The reaction conditions are shown in the following table

TABLE 2

Step1	98℃	15s
			Step2	98℃	10s
Step3	55℃	5s
			Step4	72℃	5s
		Go to Step 2x34
			Step5	72℃	1m 30s
Step6	4℃	Forever

(3) Electrophoresis

Mu.l of the sample was subjected to 1% agarose gel electrophoresis to determine whether the target band had been amplified, and the size was about 240bp (see FIG. 1).

(4) The 240bp size band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was determined.

2. IL-8 fragment and vector pGEX-4T-1 ligation transformation

Using Beijing gold sand Biotechnology Co.Ltd Uniclone one step seamless cloning kit, product number SC612

(1) Connection

Architecture configuration

TABLE 3 Table 3

Component (A)	Recombinant of interest
		2X Uniclone seamless cloning Mix	5ul
Linearization pGEX-4T-1	1ul
		IL-8 fragments	3ul
ddH ₂ O	1ul

Reaction conditions

50℃，15min

(2) Transformation

Adding 2.5ul of the product obtained in the step (1) into 50ul DH5 alpha, placing on ice for 30min, performing heat shock at 42 ℃ for 90s, placing on ice for 2min, adding 1ml of LB culture medium, culturing at 37 ℃ and 220rpm for 1h, uniformly smearing 100ul of the product on an ampicillin resistance-containing plate, and culturing at 37 ℃ in an incubator overnight.

(3) Positive clone identification

And (3) selecting the monoclonal in the step (2), carrying out colony PCR identification by using a primer IL-8F/IL-8R, identifying positive clones, carrying out further sequencing identification by a sequencing company, and retaining the clones with correct sequences.

3. IL-8 expression and purification

(1) The monoclonal is selected and added into 10ml of LB liquid medium with ampicillin resistance, cultured at 37 ℃ and 220rpm overnight,

(2) In 100ml of ampicillin-resistant blank LB liquid medium, 1% of the bacterial liquid in the step (1) is inoculated, the bacterial liquid is cultured at 37 ℃ and 220rpm overnight, and when the OD600 reaches 0.6, 0.2mmol/L of IPTG is added for induction for 4 hours.

(3) After the induction, the bacterial liquid was poured into a 50ml centrifuge tube, centrifuged at 4000rpm for 15min, the supernatant was discarded, and 10ml of a balancing liquid (140mM NaCl,2.7mMKCl,10mM Na) ₂ HPO ₄ ，1.8mM KH ₂ PO ₄ pH 7.4) the cells were resuspended. And (5) ultrasonic crushing until bacterial liquid is clarified.

(4) Transferring the crushed bacterial liquid into a centrifuge tube, centrifuging for 20min at the temperature of 15000g and 4 ℃, taking the supernatant, filtering the supernatant by a 0.45um filter, and placing the filtrate on ice.

(5) Glutathlone Beads 4FF gravity column (Hezhou Tiandi and Biotech Co., ltd., product No. SA 010005) was equilibrated with 10-fold equilibration solution, the supernatant from step (4) was added and the sample was passed through the column and repeated 4 times.

(6) The column was washed with 15-fold equilibration solution.

(7) Eluting protein with 5 times the volume of the eluent (140mM NaCl,2.7mMKCl,10mM Na) ₂ HPO ₄ ，1.8mM KH ₂ PO ₄ 10mM reduced glutathione pH 7.4).

(8) The obtained target protein was concentrated using a 10Kd ultrafiltration tube, and the buffer was replaced with 10mM BS.

(9) IL-8 assay protein concentration after concentration and SDS-PAGE detection (see FIG. 2)

2. IL-8 immunized rabbits

(1) The first immunization was carried out by mixing 500ul IL-8 (1 mg) with 500ul Freund's complete adjuvant, and injecting into 2 month old New Zealand white rabbits subcutaneously at the back.

(2) Every 2 weeks, 500ul IL-8 (1 mg) was mixed with 500ul Freund's incomplete adjuvant

New Zealand white rabbits were injected subcutaneously at back in multiple spots, 3 times at intervals.

3. Construction of phage antibody library

After the immunization of New Zealand white rabbits is finished, the animals are sacrificed, spleens are picked, monocytes are separated, total RNA is extracted, cDNA is reversely transcribed, heavy chain variable region and light chain variable region fragments are obtained through amplification by using heavy chain variable region and light chain variable region degenerate primers, antibody heavy chain variable region and light chain variable region are randomly spliced and connected with phage vectors, XL1-Blue bacteria (purchased from the company of Biotech only) are transformed, and phage antibody display libraries are assembled in the presence of auxiliary phage VSCM13 (purchased from the NTCC national collection center).

The method specifically comprises the following steps:

1. monocyte isolation

Mononuclear cells were isolated using human peripheral blood lymph isolate (LTS 1077-1, tianjin, a company of biological products technology Co., ltd.).

(1) Preparing a cell suspension: the spleen was placed on a cell sieve, placed in a culture dish with medium, gently squeezed using a 2.5mL needle syringe plunger until the spleen was completely crushed, and the cell suspension was transferred into a 15mL centrifuge tube.

(2) A15 mL centrifuge tube, 100g,10min, was filled with the cell suspension.

(3) Transferring the upper cell suspension into a 15ml centrifuge tube containing an equal volume of lymph separating liquid, carefully sucking the cell suspension by a suction tube, adding the cell suspension onto the liquid surface of the separating liquid, centrifuging for 10min at 500-1100g (1800 rpm), and accelerating for 5 and decelerating for 4.

(4) After centrifugation, the centrifuge tube is divided into four layers from top to bottom; the second layer is a cyclic milky white lymphocyte layer.

(5) The second layer of annular milky white lymphocyte layer was carefully pipetted into a new 15mL centrifuge tube, 10mL pbs was added to the resulting tube and the cells were mixed.

(6) 1800rpm, centrifugation for 10min, acceleration 5, deceleration 4.

(7) The supernatant was discarded, and 5ml of the erythrocyte lysate was added to each 15ml centrifuge tube, and the mixture was left standing at room temperature for 5 minutes after being slightly mixed.

(8) 1800rpm, centrifugation for 10min, acceleration 5, deceleration 4.

(9) The supernatant was discarded and the resulting cells resuspended in 10mL PBS.

(10) 1800rpm, centrifugation for 10min, acceleration 5, deceleration 4.

(11) The supernatant was discarded and precipitated as monocytes.

2. Total RNA extraction

The obtained monocytes were subjected to a protocol of 5X10 ⁶ Total RNA was extracted by adding 0.75ml Trizol (Thermo filter) to each cell, and then 18 parts of RNA were mixed in equal amounts by taking 1 μg of RNA each.

(1)5X10 ⁶ 0.75ml Trizol was added to each cell.

(2) Mixing the above and the below for several times, and incubating on ice for 5min to ensure the cell to be fully lysed.

(3) Centrifuge at 12000rpm for 10 minutes at 4℃and transfer the supernatant to a clean centrifuge tube.

(4) To 1ml of Trizol, 0.2ml of chloroform was added and mixed well.

(5) Incubating on ice for 2-3min.

(6) Centrifuge at 12000rpm at 4℃for 15min.

(7) Transferring the supernatant phase to a clean centrifuge tube.

(8) Adding isopropanol with equal volume into the supernatant and uniformly mixing.

(9) Incubate for 10min at room temperature, centrifuge at 12000rpm at 4℃for 10-15 min.

(10) The supernatant was discarded and the pellet was retained.

(11) The precipitate was washed with 1ml of 75% ethanol.

(12) Centrifuge at 12000rpm at 4℃for 5min.

(13) The supernatant was discarded as much as possible.

(15) Drying at room temperature for 5-10min, wherein no liquid is visible on the tube wall.

(16) Add 20-50. Mu.l of RNAse free water-soluble RNA.

(17) 1. Mu.l of RNA was used for electrophoresis detection and RNA concentration measurement.

(18) The RNA is packaged and stored in a refrigerator at the temperature of-80 ℃ to avoid repeated freezing and thawing.

3. cDNA Synthesis

Mu.g of RNA was taken and used with cDNA synthesis kit (K1612, thermo filter).

(1) cDNA was synthesized by taking 5. Mu.g of RNA.

(2) 10 μl of RNA/primer mix μre system is shown in Table 4 below, comprising:

TABLE 4 Table 4

System components	Volume of
		RNA	5μg
50μM Oligo(dT) ₂₀	1μl
		Hex Random Primer	1μl
10mM dNTP	1μl
		DEPC-treated water	Total volume of 10. Mu.l

(3) Incubate at 65℃for 5min, place on ice for 1min.

(4) Other reagents were added as shown in Table 5 below to prepare cDNA Synthesis Mix for the next reaction.

TABLE 5

System components	1 reaction
		10xRT buffer	2μl
25mM MgCl ₂	4μl
		0.1M DTT	2μl
RNaseOΜT(40Μ/μl)	1μl
		Superscript III RT(200Μ/μl)	1μl

(5) Add 10. Mu.l of cDNA Synthesis Mix to RNA/primer mix gently, centrifuge, and react under the following Table 6 conditions:

TABLE 6

Step1	50℃	50min
			Step2	85℃	5min
		Cooling on ice, and briefly centrifuging
			Step3	RNaseH	1μl
Step4	37℃	20min

(6) The cDNA product was stored in aliquots at-20 ℃.

4. Antibody heavy chain variable region (VH) and light chain variable region (VL) amplification

The synthesized cDNA was diluted 10-fold and used as a template for amplifying the heavy and light chain variable regions.

(1) The system was formulated as shown in Table 7 below, taking VH as an example:

TABLE 7

(2) The reaction conditions are shown in the following table

TABLE 8

(3) Electrophoresis

Mu.l of the sample was subjected to 1% agarose gel electrophoresis to determine whether the target band had been amplified, and the size was about 400bp (see FIG. 3).

(4) All amplified VH were mixed in equal amounts, added with 0.1 volumes of 3M sodium acetate and 2.2 volumes of ethanol, mixed well and left at-20 ℃ overnight.

(5) Centrifugation at 16000g at 4℃for 15min, removal of supernatant, rinsing with 1ml of 70% ethanol (room temperature), drying at room temperature, adding 200. Mu.l of water for dissolution back and gel electrophoresis with 1% gel.

(6) The 400bp size band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was determined.

(7) The amplification and recovery of VL was performed according to steps (1) to (6), and the results are shown in FIG. 4.

(8) The concentration of nucleic acid after purification of VH and VL was adjusted to 100 ng/. Mu.l, and stored at-20 ℃.

5. Amplification of Cκ -pelB

(1) Cκ -pelB and CL-pelB were amplified using vectors pCκ and pCL (vectors purchased from add gene) (100 ng/. Mu.l) as templates

For example, amplified C.kappa. -pelB is shown in Table 9.

TABLE 9

pCκ	10μl
		HCK (see the aforementioned references for sequences)	60μl
Pelb (see the foregoing references for sequences)	60μl
		ddH ₂ O	370μl
PrimerSTAR	500μl
		Total	1000μl

(2) The reaction conditions are shown in Table 10

Table 10

(3) Mu.l of the product was taken and electrophoretically detected, and the band size was about 400bp.

(4) All amplified Cκ -pelB were mixed, added with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol, mixed well and left at-20℃overnight.

(5) Centrifugation at 16000g at 4℃for 15min, removal of supernatant, rinsing with 1ml of 70% ethanol (room temperature), drying at room temperature, adding 200. Mu.l of water for dissolution back and gel electrophoresis using 1% gel.

(7) The purified C kappa-pelB was diluted to a final concentration of 100 ng/. Mu.l and stored at-20 ℃.

(8) Amplifying CL-pelB, and performing the operation according to the steps (1) - (7), wherein the amplified template pCκ is replaced by pCL.

6. Rab V kappa/Rab C kappa/CL/Rab VH fusion

(1) The VL, VH, ck-pelB were mixed in varying amounts as shown in Table 11 below

VL concentration 100 ng/. Mu.l, VH concentration 100 ng/. Mu.l, C kappa-pelB concentration 100 ng/. Mu.l

TABLE 11

VL	10μl
		VH	10μl
Ck-pelB	10μl
		Primerstar Mix	500μl
H ₂ O	470μl
		Total	1000μl

(2) The reaction conditions are shown in Table 12

Table 12

(3) Amplification of Rab VL/Rab C kappa/Rab VH, see Table 13

TABLE 13

Splice product	50μl
		C-5' SFIVL (see the abovementioned references for sequences)	4μl
c-3' sfivh (see the aforementioned references for sequences)	4μl
		ddH ₂ O	17μl
PrimerSTAR	25μl
		Total	100μl

(4) Amplification conditions are shown in Table 14

TABLE 14

Step1	98℃	15s
			Step2	98℃	10s
Step3	55℃	5s
			Step4	72℃	5s(Go to Step2 x 39)
Step5	72℃	1m 30s
			Step6	4℃	Forever

(5) Electrophoresis detection

10 mu l of PCR product is taken, electrophoresis detection is carried out, and the band size of the target gene after fusion is 1.2Kb. (see FIG. 5)

(6) All amplified Rab VL/Rab ck/Rab VH, 0.1 volumes of 3M sodium acetate and 2.2 volumes of ethanol were added, mixed well and left overnight at-20 ℃.

(7) Centrifugation at 16000g at 4℃for 15min, removal of supernatant, rinsing with 1ml of 70% ethanol (room temperature), drying at room temperature, adding 200. Mu.l of water for dissolution back and gel electrophoresis using 1% gel.

(8) The 1.2Kb size band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was determined.

(9) The Rab VL/Rab C kappa/Rab VH purified nucleic acid was diluted to a final concentration of 150 ng/. Mu.l and stored at-20 ℃.

(10) The fusion of rabV lambda/rabCL/Rab VH is performed according to steps (1) - (9).

7. SfiI cleave Rab V kappa/Rab C kappa/CL/Rab VH and RabV lambda/RabCL/Rab VH

Take Rab V kappa/Rab C kappa/CL/Rab VH cleavage as an example

(1) The enzyme digestion system is shown in Table 15

TABLE 15

human Vκ/human Cκ/CL/human VH(150ng/μl)	200μl
		10Xbuffer	30μl
H ₂ O	60μl
		SfiI 40u/μl	10μl

(2) The reaction is carried out for 3 hours in a water bath at 50 ℃.

(3) Electrophoresis was performed using a 1% gel, and a 1.2 kb-sized band was excised and recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706).

(4) After SfiI digestion is detected, rab VL/Rab Ck/Rab VH fragments are recovered, the concentration is adjusted to 50 ng/. Mu.l, and the mixture is preserved at-20 ℃.

(5) Cleavage of Rab V lambda/Rab CL/Rab VH, is performed according to steps (1) - (4).

8. SfiI cleavage pC3C (vector from add gene)

(1) The enzyme digestion system is shown in Table 16

Table 16

pC3C 1μg/μl	50μl
		10Xbuffer	30μl
H ₂ O	208μl
		SfiI 40u/μl	12μl

(2) The reaction is carried out for 3 hours in a water bath at 50 ℃.

(3) Electrophoresis using a 1% gel will cut two bands, a 3.5Kb and a 1.2Kb sized fragment.

(4) The vector backbone of 3.5Kb was recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706).

(5) The concentration of the recovered vector backbone after Sfi cleavage was adjusted to 100 ng/. Mu.l.

9. Connecting pC3C (SfiI) and Rab VL/human C kappa/Rab VH (SfiI)

(1) The connection system is shown in Table 17

TABLE 17

pC3C after cleavage of 100 ng/. Mu.l SfiI	1.5μl
		50 ng/. Mu.l SfiI post-digestion human VL/human C kappa/human VH	2μl
10XT4 DNA ligase buffer	2μl
		H ₂ O	13.5μl
T4 DNA ligase 2000u/μl	1μl

The control group is shown in Table 18

TABLE 18

pC3C after cleavage of 100 ng/. Mu.l SfiI	1.5μl
		10XT4 DNA ligase buffer	2μl
H ₂ O	15.5μl
		T4 DNA ligase 2000u/μl	1μl

(2) The reaction was carried out at 16℃overnight.

(3) All ligation reactions were combined and 1/10 volume of 3M sodium acetate solution was added.

(4) Adding 2.2 times of pre-cooled absolute ethyl alcohol, uniformly mixing up and down, and precipitating at-20 ℃ overnight.

(5) 16000g, centrifuged at 4℃for 30min and the supernatant carefully discarded.

(6) The precipitate was gently washed with 70% ethanol, 16000g, centrifuged at 4℃for 5min and the supernatant discarded.

(7) And (5) drying at room temperature.

(8) The reaction is dissolved with an appropriate amount of water, usually 1 ligation reaction is performed with a volume of 1. Mu.l ddH ₂ O is fully dissolved and is preserved at the temperature of minus 20 ℃.

(9) The connections pC3C (SfiI) and Rab V lambda/Rab CL/Rab VH are operated according to steps (1) - (8).

10. Ligation product conversion XL1-Blue

(1) XL1-Blue electric shock competent ice was placed for 10min for dissolution.

(2) Taking 19 μl of the precipitated and concentrated connection product, adding into a 1.5ml centrifuge tube, ice-bathing, adding 300 μl of XL1-Blue electric shock competence, mixing, rapidly transferring into a 2mm electric shock cup, and standing on ice for 1min.

(3) Electric shock conditions: 2.5KV,4ms

(4) After the completion of the electrotransfer, 5ml (1 ml+2ml+2ml) of SOC medium was rapidly added, and the mixture was transferred to a 50ml tip centrifuge tube, and incubated at 37℃and 250rpm for 1 hour. Mu.l of the bacterial liquid was added to 198. Mu.l of LB and simultaneously uniformly spread on LB plates containing 100. Mu.g/. Mu.l of carbenicillin, and incubated overnight at 37℃for calculation of transformation efficiency and colony PCR identification positive rate. The PCR results are shown in FIG. 6.

(5) 10ml SB medium, 3. Mu.l 100. Mu.g/. Mu.l carbenicillin, and 30. Mu.l 5. Mu.g/. Mu.l tetracycline were added. The culture was carried out at 37℃and 250rpm for 1 hour.

(6) Add 4.5. Mu.l 100. Mu.g/. Mu.l carbenicillin and continue at 37℃at 250rpm for 1-4h.

(7) The cultured bacteria were transferred to a 500ml flask, 84ml SB medium, 42.5. Mu.l 100. Mu.g/. Mu.l carbenicillin, 170. Mu.l 5. Mu.g/. Mu.l tetracycline, and 1ml VCM 13 helper phage (10) ¹¹ -10 ¹² pfu/ml)，37℃，275rpm，90min。

(8) Mu.l of 50. Mu.g/. Mu.l kanamycin was added, incubated at 37℃and 275rpm overnight.

(9) 3000g, and centrifuged at 4℃for 15min.

(10) Precipitation phage

The supernatant (200 ml) was transferred to a 500ml clean centrifuge tube, 8g PEG-8000 and 6g NaCl were added, and the mixture was placed at 37℃at 300rpm for 5min to promote dissolution. Placing on ice for 30min-1h.15000g, centrifugation at 4℃for 15min. The supernatant was discarded, the centrifuge flask was placed upside down on filter paper and left to dry for 10min, taking care to remove excess liquid. Phage were resuspended in 2ml TBS (pipetted up and down) with 1% BSA, centrifuged at 16000g at 4℃for 5min, and the supernatant was passed through a 0.22 μm filter (Millipore) and transferred to a 2ml centrifuge tube. The product can be stored directly on ice in a short time or stored at-20deg.C by adding 0.01 times of 2% sodium azide in volume and adding 1 time of glycerol in a long time.

2. Phage antibody library screening exemplified by IL-8 phage antibody screening

5 rounds of screening were performed using 96-well plates coated with IL-8 protein, and then monoclonal antibodies were selected for identification of IL-8 specific antibodies.

1. Antigen coating

(1) IL-8 was added to a carbonic acid buffer at a concentration of 2. Mu.g/ml,

(2) Kang Ninggao protein adsorption ELISA 96-well plate, adding 50 mu lIL-8 into each well, and standing at 4deg.C overnight;

(3) Discarding the supernatant, adding 200 μl/well of 5% skimmed milk powder, and standing at 37deg.C for 1 hr;

(4) The supernatant was discarded, and washed 5 times with 0.05% TBST.

2. IL-8 antibody screening

(1) Phage antibody library was diluted 10-fold with 2% skimmed milk powder, 2 96-well microwells coated with IL-8 antigen were taken, 100 μl of diluted phage was added, and left at 37℃for 1h.

(2) The supernatant was discarded, washed 5 times with 0.05% TBST, 100. Mu.l of 100mM glycine was added, and the mixture was left at 37℃for 15 minutes, during which time the blow was continued, and 9. Mu.l of 1M Tris was added.

(3) The phage eluted in step (2) was added to 2ml XL1-Blue bacteria and left at room temperature for 15min.

(4) 6ml of SB medium was added, 1.6. Mu.l of 100. Mu.g/. Mu.l of carbenicillin, 12. Mu.g of 5. Mu.g/. Mu.l of tetracycline were added, and the mixture was incubated at 37℃for 1 hour at 250 rpm.

(5) 2.4. Mu.l of 100. Mu.g/. Mu.l of carbenicillin were added and the culture was continued for 1h;

(6) 1ml of helper phage VCSM13 (10) ¹¹ -10 ¹² pfu/ml), transferred to a 500ml flask, added 91ml SB medium, added 46. Mu.l 100. Mu.g/. Mu.l carbenicillin, 184. Mu.l 5. Mu.g/. Mu.l tetracycline, incubated at 37℃for 1.5h at 250rpm, added 140. Mu.l 50. Mu.g/. Mu.l kanamycin, incubated at 37℃overnight at 250 rpm.

(7) Phage antibody concentration: centrifuging the bacterial liquid in the last step to remove bacterial bodies, and collecting supernatant (100 ml); 4g PEG-8000 and 3g NaCl were added and the mixture was placed at 37℃at 300rpm for 5min to promote dissolution. Placing on ice for 30min-1h.15000g, centrifugation at 4℃for 15min. The supernatant was discarded, the centrifuge flask was placed upside down on filter paper and left to dry for 10min, taking care to remove excess liquid. Phage were resuspended in 2ml TBS (pipetted up and down) with 1% BSA, centrifuged at 16000g at 4℃for 5min, and the supernatant was passed through a 0.22 μm filter (Millipore) and transferred to a 2ml centrifuge tube. The product can be stored directly on ice in a short time or stored at-20deg.C by adding 0.01 times of 2% sodium azide in volume and adding 1 time of glycerol in a long time.

(8) Taking phage of step (7), and carrying out 5 rounds of screening according to steps (1) - (7).

(9) ELISA identification of antibody libraries from five rounds of screening:

1) ELISA 96-well plates, coated with 100ng of IL-8 per well;

2) 100 μl phage (diluted 10-fold with 2% skimmed milk powder) was added and incubated at 37deg.C for 1h;

3) 0.05% TBST wash 5 times;

4) 100 μl of anti-M13-HRP antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) diluted 2000-fold with 5% skimmed milk powder was added and incubated at 37deg.C for 1h;

5) 0.05% TBST wash 5 times;

6) Adding 100 μl TMB, and developing for 5min in dark;

7) Add 50 μl1M H ₂ SO ₄ The reaction was terminated.

8) Absorbance was measured at 450nm using a microplate reader.

The results are shown in FIG. 7 and Table 19.

Table 19 ELISA identification of phage library from round 5 screen

3. IL-8 monoclonal antibody identification

(1) Taking IL-8 phage of 5 th round of screening, diluting 10 ^-7 1 μl was added to 200 μl XL1-Blue bacteria, and left at room temperature for 15min;

(2) Coating all the bacteria in the step (1) on a carbenicillin LB individual culture medium containing 100 mug/ml, and culturing overnight at 37 ℃;

(3) 200 single clones were selected and added to 6ml of liquid LB medium containing 10. Mu.g/ml tetracycline and 100. Mu.g/ml carbenicillin, and cultured at 37℃and 250rpm for 4 hours, a small amount of bacterial liquid was taken out, and bacterial liquid PCR identification was performed to select clones capable of amplifying a band size of 1200 bp.

Colony PCR amplification system is shown in Table 20:

table 20

Bacterial liquid	0.5μl
		VHSEQ (sequence see the aforementioned references)	0.2μl
vlseq (see the aforementioned references for sequences)	0.2μl
		ddH ₂ O	4.1μl
PrimerSTAR	5μl
		Total	10μl

Colony PCR reaction conditions are shown in Table 21:

table 21

(4) The positive clone broth from step (3) was divided evenly into 2 parts, one part was added to 3. Mu.l of helper phage VCSM13 (10) ¹¹ -10 ¹² pfu/ml), left at room temperature for 20min, 2.1. Mu.l of 50. Mu.g/. Mu.l kanamycin was added, and the other aliquot was incubated overnight at 37℃at 250rpm without any additional material;

(5) Centrifuging the bacteria containing helper phage at 4000rpm for 10min, transferring the supernatant into a clean centrifuge tube, diluting the supernatant 10 times, adding 100 μl of the supernatant into an ELISA plate coated with IL-8, incubating at 37deg.C for 1H, washing with 0.05% TBST 5 times, adding 2000 times diluted anti-phage HRP-labeled antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., ltd.), incubating at 37deg.C for 1H, washing with 0.05% TBST 5 times, adding 100 μl TMB, developing for 2min, adding 50 μl1M H ₂ SO ₄ The reaction was terminated and absorbance was measured at 450nm using a microplate reader and was 2.1 times higher than the control as positive clones.

(6) Selecting positive clones in the step (5), extracting plasmids without adding bacteria of auxiliary phage, and sequencing to obtain heavy chain variable region and light chain variable region sequences.

3. Expression and Activity characterization of fully humanized antibodies

1. Fully humanized vector construction

(1) Designing corresponding primers according to the obtained antibody heavy chain variable region or light chain variable region sequence, respectively amplifying, adding secretion signal peptides at the N ends of the heavy chain and the light chain, and adding an Fc fragment of IgG1 at the C end of the heavy chain;

(2) The heavy chain variable region and the light chain variable region were each homologously recombined into the pcDNA3.4 vector. (see FIG. 8) (pcDNA3.4 vector available from Wohan vast, biotechnology Co., ltd.)

2. Expression of fully humanized antibodies

(1) Extracting the corresponding heavy chain expression vector and light chain expression vector respectively by using endotoxin removal kit (purchased from OMEGA);

(2) Transfection with PEI was performed when 293T cells (purchased from the Living technologies Co., ltd.) reached 80% confluence;

(3) 12h after transfection, the cells were replaced with serum-free protein expression medium at 37℃and 5% CO ₂ Culturing for 7 days;

(4) Antibodies were purified using protein A/G packing. The results are shown in FIG. 9.

3. Identification of fully humanized antibody Activity

Taking IL-8 antibody Activity assay as an example

(1) ELISA identification

The obtained antibody was adjusted to a concentration of 1mg/ml, diluted 2000-fold with 5% nonfat milk powder, added to an ELISA well coated with IL-8 antigen, incubated at 37℃for 1H, washed 5 times with 0.05% TBST, diluted 2000-fold with 5% nonfat milk powder against human Fab-HRP secondary antibody (available from Beijing Soy Biotechnology Co., ltd.), incubated at 37℃for 1H, washed 5 times with 0.05% TBST, added 100. Mu.l TMB, developed for 2min, and added 50. Mu.l of 1M H ₂ SO ₄ The reaction was terminated and absorbance was measured at 450nm using a microplate reader. The results are shown in Table 22.

Table 22 ELISA identification of purified YX20 binding to IL-8

(2) The IL-8 protein is detected by using YX20 as an immobilized antibody and YX5, YX20 and YX40 as detection antibodies in combination as follows:

ELISA sandwich method is adopted for detection, a monoclonal fully humanized antibody YX20 is coated on an ELISA plate, antigen IL-8 is added, screened YX5, YX20 and YX40 biotin labeled antibodies (biotin brand: thermo, product number: VF 300852) are added, different combinations are carried out, and streptavidin (brand: thermo Fisher, product number: VE 302151) with HRP label is added, so that detection color development is carried out, the darker color indicates the stronger capturing capability on the IL-8 antigen, and meanwhile, the pair of antibodies are also indicated to be suitable for detection of the IL-8 antigen. The blank control refers to free phase without antibody and with HRP-labeled streptavidin alone.

The specific operation is as follows:

1) ELISA 96-well plates, coated with 100ng of YX20 antibody per well;

2) Mu.l IL-8 (250 ng/ml) was added and incubated for 1h at 37 ℃;

3) 0.05% TBST wash 5 times;

4) Adding biotin-labeled antibodies YX5, YX20 and YX40 diluted 2000 times with 5% skimmed milk powder, and incubating at 37 ℃ for 1h;

5) 0.05% TBST wash 5 times;

6) Adding 5% skimmed milk powder to dilute 5000 times of streptavidin marked with HRP, and incubating for 10min at room temperature;

7) 0.05% TBST wash 5 times;

8) Adding 100 μl TMB, and developing for 5min in dark;

9) Add 50 μl1M H ₂ SO ₄ The reaction was terminated.

10 Absorbance was measured at 450nm using a microplate reader.

The results are shown in Table 23.

Table 23 Sandwich method for identifying antibody pairs for detection of IL8

Note that: column coating phase, row detection phase, OD450 absorbance.

(3) Neutrophil chemotaxis inhibition

1) Isolation of neutrophils from human peripheral blood (Tianjin ocean, TBD LZS 11131), and adjustment of neutrophil concentration to 5X10 Using 1640 ⁶ Individual/ml;

2) Using a 24-well Trans-well plate (Corning Costar, 3415), pore size 3um, the experiments were divided into 3 groups: blank group, lower chamber added with 400ul blank 1640; positive control, lower chamber with 400ul 1640 containing 10nMIL 8; in the experimental group, 400ul 1640 containing IL8 at a concentration of 10nM and IL8 antibody at 5ug/ml was added to the lower chamber; all 3 groups, the upper chamber was charged with 200ul (concentration 5X10 ⁶ Individual/ml) neutrophils;

3)5％ CO ₂ the cells were left at 37℃for 2 hours, the upper chamber was discarded, and the cell count in the lower chamber was calculated by a cytometer.

The results are shown in Table 24 and Table 25, FIG. 10.

Table 24 anti-IL 8 antibodies inhibit neutrophil chemotaxis for IL8

Table 25 inhibition of neutrophil chemotaxis by anti-IL 8 antibodies (unit:%)

	Compound hole 1	Multiple hole 2	Multiple hole 3
				YX20	100	103.3582102	110.0746306

(4) anti-IL 8 antibodies against tumor inhibition

1) Nude mice were divided into 2 groups, 3 groups and control group, and 100ul of 1X10 was injected subcutaneously into the back of each group before the start of the experiment ⁶ HCT116 tumor cells (colon tumor cells);

2) After 3 days, 200ul of PBS was injected intraperitoneally into each nude mouse in the control group, 200ul of PBS containing 500ug of anti-IL 8 antibody was injected intraperitoneally into each nude mouse in the experimental group, and every 3 days;

3) After 4 rounds of injection, the last day of injection, nude mice were sacrificed, tumors were removed and weighed. The results are shown in Table 26 and FIG. 11.

Table 26 anti-IL 8 antibodies against tumor inhibition (unit: g)

Numbering device	1	2	3
				control	0.27	0.26	0.3
YX20	0.06	0.03	0.19

Claims

An il-8 antibody, characterized by the following heavy and light chain CDR amino acid sequences, which are monoclonal antibody YX 20:

YX20 heavy chain CDR sequence:

CDRH1 amino acid sequence GFSLNNYA

CDRH2 amino acid sequence VGSDDIP

CDRH3 amino acid sequence ASGYVGDDRYNI;

YX20 light chain CDR sequence:

CDRL1 amino acid sequence PSVYNNY

CDRL2 amino acid sequence AAS

CDRL3 amino acid sequence AGAYSNDSDDG.
2. The IL-8 antibody of claim 1, wherein the heavy and light chain variable region amino acid sequences of the monoclonal antibody are as follows:

YX20 heavy chain variable region amino acid sequence:

GSTGDQEQLVESGGRLVTPGTPLTLTCTVSGFSLNNYAMSWVRQAPGKGLEYIGVVGSDDIPFYASWAKGRFAISKTSTTVDLKITSPTTEDTATYFCASGYVGDDRYNIWGPGTLVTISS；

YX20 light chain variable region amino acid sequence:

GSTGDAAVVLTQTPSPVSAAVGGTVSISCQSSPSVYNNNYLSWYQQKPGQPPKLLIYAASTLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGAYSNDSDDGFGGGTKLEIK。
3. the IL-8 antibody of claim 1, wherein the antibody includes, but is not limited to: immunoglobulin, fab, (Fab') 2 or single chain antibody.
4. Use of an IL-8 antibody according to any one of claims 1-3 for the preparation of a formulation for the detection of IL-8.
5. Use of an IL-8 antibody according to any one of claims 1-3 for the preparation of a formulation for the treatment of colon tumors.