CN108218998A - A kind of Fc segments of saltant type humanized IgG and preparation method and application - Google Patents

Abstract

The invention discloses a kind of Fc segments of saltant type humanized IgG and preparation method and application.In CH2 the and CH3 structural domains of Fc segments a pair of of disulfide bond is introduced respectively, and then put forward high molecular stability and resistant to aggregation ability.For the Fc segments of saltant type humanized IgG relative to the Fc of wild type, stability is more preferable, can reduce the production of monoclonal antibody or Fc fusion proteins, purifying, preserve cost；Its resistant to aggregation ability is more preferable, and can reduce leads to Clinical practice risk because of the aggregation of albumen.

Description

A kind of Fc segments of saltant type humanized IgG and preparation method and application

Technical field

The present invention relates to biotechnology, in particular to a kind of Fc segments of saltant type humanized IgG and preparation method thereof With application.

Background technology

Antibody is widely used in every field, especially life science and clinic with its high specificity, high sensitivity It treats such as ELISA, Western blot, immunofluorescence analysis, disease quick diagnosis and biological agent is used as to treat various diseases Disease.Therefore, since the 21th century, people also begin to increasingly pay close attention to the research and development and clinical practice of therapeutic antibodies drug, this It is because it is small to human body toxic side effect, has the effect of natural and high degree of specificity, and has createed huge society's effect Benefit and economic benefit.

Meanwhile the fusion protein based on Fc segments not only with its molecular weight it is relatively small and with good tissue infiltration Property, and the Fc segments being coupled thus can extend the blood plasma half of drug with the effects interaction of molecules such as Fc receptors (FcRn) Decline phase and inducible ADCC, CDC effect or phagocytosis.

However, these antibody drugs and Fc fusion proteins, as bioactive macromolecule, it also has the limitation of itself, If stability is poor, resistant to aggregation ability is weak, during its expression, purifying, storage etc., can tend to assemble or degrade.And egg Immunogenicity caused by white aggregation, not only reduces drug effect, while very big risk is also brought to clinical practice.Therefore, it carries Highly resistance body heat stability and resistant to aggregation ability are one of the field urgent problems to be solved.

Invention content

It is an object of the invention to overcome the defects of Fc segments stability is poor, resistant to aggregation ability is weak in the prior art, provide A kind of Fc segments of saltant type humanized IgG and preparation method and application, compared with wild type Fc, stability and resistant to aggregation energy Power is improved, and is of great significance in the research and development, production, clinical practice of antibody or Fc fusion proteins.

To achieve the above object, it is by Ren Yuan the present invention provides a kind of Fc segments of the saltant type humanized IgG It is obtained after introducing disulfide bond in the CH2 structural domains of the Fc segments of IgG and CH3 structural domains.

In said program preferably, the 242nd in the amino acid sequence of the Fc segments of the saltant type humanized IgG the, the 334th Position, the 343rd and the 431st amino acid for cysteine, (amino acid position number is referring to http://www.imgt.org/ Eu numbering in IMGTScientificChart/Numbering/Hu_IGHG nber.html, the same below).

Preferably, the Fc segments of the saltant type humanized IgG, are named as S23, and amino acid full length sequence is Seq ID No.1。

Optionally, the coding gene sequence of the Fc segments of the saltant type humanized IgG is Seq ID No.2.

The present invention also provides the preparation methods of the Fc segments of above-mentioned saltant type humanized IgG, and its step are as follows：

(1) gene of the Fc segments of humanized IgG is obtained；

(2) design primer amplifies to obtain by the designated amino acid base mutation in Fc into the base of encoding aminothiopropionic acid Saltant type Fc；

(3) carrier for expression of eukaryon being built with saltant type Fc gene orders, transfection mammalian cell carries out eukaryotic expression, Purifying obtains target Fc segments.

Present invention further teaches above-mentioned saltant type humanized IgG application of the Fc segments in Fc fusion proteins are prepared and Prepare the application in monoclonal antibody.

The present invention by the methods of CD, molecular sieve, fluorescence detector, ultraviolet specrophotometer, ELISA to the two level of S23 Structure, existence form, stability, resistance to aggregation are identified, are compareed with wild type Fc.Related experiment also turns out S23 It can be combined with Fc receptors, therefore, modify while improved S23 improves itself physicochemical property and do not destroy itself Biological effect.

Beneficial effects of the present invention：

1) relative to the Fc of wild type, stability is more preferable, can reduce monoclonal antibody or Fc fusion proteins production, Purifying preserves cost；

2) relative to the Fc of wild type, resistant to aggregation ability is more preferable, and can reduce causes clinic to make because of the aggregation of albumen Use risk.

Description of the drawings

Fig. 1 is protein immunoblot detection figure after the expression and purification of Fc, S23 albumen.

Fig. 2 is SDS electrophoretograms after the expression and purification of Fc, S23 albumen.

Fig. 3 is the existence form analysis chart (monomer, dimer etc.) of Fc, S23 protein molecular.

Fig. 4 is the analysis chart of C.D analysis Fc, S23 molecular conformation.

Fig. 5 compares figure for stability of Fc, S23 albumen under denaturant conditions.

Fig. 6 compares figure for Fc, S23 albumen thermal stability.

Fig. 7 compares figure for Fc, S23 albumen resistance to aggregation.

Fig. 8 compares figure for protein molecular existence form in the experiment of Fc, S23 albumen resistance to aggregation.

Fig. 9 is the streaming figure after Fc, S23 albumen are combined with Fc receptors (Fc γ RI).

Specific embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.Following embodiment is with this Implemented under premised on inventive technique scheme, give detailed embodiment and specific operating process, but the present invention Protection domain is not limited to following embodiments.

Embodiment 1：Expand Fc, S23 fragment gene

People source blood sample is taken, cell total rna, obtained cell are extracted with Trizol Reagent (Invitrogen companies) Total serum IgE is dissolved in 50 water of the μ L without RNA enzyme.Then M-MLV Reverse Transcriptase kits (Promega companies) are used, using random Primer, reverse transcription obtain cDNA.

According to the gene order (GenBank of the Fc of IgG:KJ905798.1), its amino acid sequence is analyzed, designs Fc genes Forward and reverse primer amplification target fragment：Forward primer 5-GACGCGGCCCAGCC G GCCGCACCTGAA CTC CTGGGGGGA C-3, (horizontal line is labeled as Sfi I restriction enzyme sites)；Reverse primer 5-GCCCTC C TCGAG TCA TTT ACC CGG AGA CAG GGA G-3, (horizontal line is labeled as Xho I restriction enzyme sites).First with forward primer and reverse primer using cDNA as Template carries out PCR, obtains Fc genetic fragments, recycles point mutation kit by four amino acid mutations of Fc segments into half Guang ammonia Acid obtains S23 genetic fragments (gene order is Seq ID No.2, and amino acid sequence is Seq ID No.1).

Agarose gel electrophoresis recycles Fc, S23 genetic fragment, with Sfi I and Xho I digestion Fc, S23 genes and carrier Fc, S23 gene are connect, Transformed E .coli Top10 competence, then by Psectag2A after glue recycling with carrier Psectag2A Picking monoclonal send sequencing.According to sequencing result, the correct monoclonal of picking sequence is used for subsequent experimental.

Embodiment 2:It expresses and purifies Fc, S23

By each 40ug of Fc, S23 plasmid using PEI (2mg/mL PEI) (Polyethylenimine " Max ", (Mw 40, 000)-High Potency Linear PEI,Polysciences,Inc.,Catalog No.:24765-2,2g) transfect 293F cells, respectively with 293F expression culture medium (invitrogen) 40ml in 37 DEG C, 150rpm shaking table cultures 6 days.

Then, centrifuge (6000g, 4 DEG C, 15min) collecting 293F cell conditioned mediums, (supernatant is anti-using mouse anti-human igg Fc as first The sheep anti-mouse igg that body, HRP are marked is that secondary antibody is to carry out protein immunoblot detection, and the results are shown in Figure 1, swimming lane 1：Marker； Swimming lane 2：Fc cell conditioned mediums；Swimming lane 3：S23 cell conditioned mediums.The expression of clearly visible Fc, S23 albumen from figure), by the upper of collection After clear filtering, Fc, S23 albumen are purified with Protein G fillers (GE companies).Then with the ultrafiltration that molecular cut off is 10kD from The destination protein is concentrated by ultrafiltration in heart pipe (Merck Millipore companies), its purity is verified through SDS-PAGE, as a result such as Fig. 2 institutes Show, swimming lane 2 is Fc segments, swimming lane 3 is S23 segments.

Embodiment 3：Fc, S23 existence form and molecular conformation

AKTA analyzes Fc, S23 existence form：By Fc, S23 protein concentration after purification to 1mg/ml, PBS (pH7.4) is Elution buffer crosses 10/300 GL of Column Superdex 75Increase, wherein, flow velocity 0.5ml/min, so it is right Its existence form is detected, and such as Fig. 3, reference standard curve is it can be found that the two molecular weight of albumen is about 56kDa, as a result Show that they exist with dimeric forms.

CD detects protein molecular conformation：Fc, S23 albumen are diluted to 50ug/ml, PBS (pH7.4) is control, near purple Its circular dichroism is detected under the conditions of outer end (wavelength X=190nm~260nm) different wave length, and then analyzes Protein secondary structure, is led to 10/300 GL of Column Superdex 75Increase (GE companies) are crossed to be analyzed, reference standard curve it can be found that The two molecular weight of albumen is about 56kDa, and the results are shown in Figure 4, and there are one apparent troughs at λ=218nm, show above-mentioned egg White secondary structure is beta sheet.

Embodiment 4：Fc, S23 stability are compared with resistance to aggregation

Denaturant conditions stability inferior is analyzed：Configuration concentration is the urea liquid of 0M to 10M, and concentration gradient 0.5M is common 21 samples；Then Fc, S23 albumen are added in ambient temperature overnight in the urea liquid of various concentration gradient handle, volume is 200ul, Fc, S23 final concentration of protein are 100ug/ml.Then, it is excited in 280nm with fluorescence detection equipment, is detected at 340nm Each fluorescent intensity, as a result such as Fig. 5, it can be found that compared to Fc from figure, the fluorescence signal downward trend of S23 is slower, Show that S23 more stablizes under denaturant conditions.

Thermal stability analysis：Compare Fc, S23 albumen (50ug/ml) thermal stability using circular dichroism spectrometer, PBS solution is Blank control, set temperature gradient condition be per 2min increase 1 DEG C, terminate reaction temperature be 94 DEG C, per 30s wavelength X= It is detected at 218nm and records data, as shown in fig. 6, analysis detects the molar absorptivity of its left and right circularly polarized light at different temperatures The difference of coefficient, and then compare its thermal stability difference, it can be found that the thermal stability for modifying improved S23 is more much higher than Fc.

Resistance to aggregation is analyzed：

A) Fc, S23 final concentration of protein are adjusted to 2mg/ml, handle in 60 DEG C of water-baths, sample at regular intervals, Absorbance is detected at λ=320nm and records data, wherein, PBS solution is blank control, compares the resistant to aggregation of Fc, S23 albumen Property it is strong and weak, it can be found that S23 signals ascendant trend is relatively slow from Fig. 7, show that its resistant to aggregation ability is more preferable.

B) Fc, S23 protein sample that 60 degree of water-baths are handled to 551 hours pass through 13000rpm, and 4 DEG C, 15min is centrifuged, Supernatant is taken to cross 75 Increase 10/300GL of Column Superdex, wherein, flow velocity 0.5ml/min, and then it is deposited It is detected in form.As a result such as Fig. 8, analysis are found, two peaks of the apparent appearance of Fc albumen at this time, an aggregation peak, one two Aggressiveness peak, and then only there are one uniform dimer peaks by S23, as existence form during firm expression and purification, illustrate that albumen does not have Occur aggregation.

Embodiment 5：The combination of Flow cytometry Fc, S23 and Fc γ RI

U937 cell surfaces can express Fc γ RI, therefore compare Fc, S23 albumen and Fc γ RI parents with Flow cytometry experiments And power.Fc, S23 albumen and U937 cells (U937 cell surfaces can express Fc γ RI) of gradient dilution are incubated 1 hour at 4 degree. Unbonded albumen is washed off with culture medium.By the use of the goat anti-human igg Fc antibody (Sigma-Aldrich) of fluorescent marker as secondary antibody with Cell is incubated 1 hour at 4 degree.It is cleaned with 1%PBSA and cell is resuspended, then sample is positioned on flow cytometer (Becton Dickinson its binding force) is measured.Statistics indicate that, the S23 by modification transformation still keeps being combined work with Fc γ RI in Fig. 9 Property.

Sequence table

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<120>A kind of Fc segments of saltant type humanized IgG and preparation method and application

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Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

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Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

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Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

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Ser Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

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gcacctgaac tcctgggggg accgtcagtc ttctgcttcc ccccaaaacc caaggacacc 60

ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120

cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180

ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240

caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 300

cccatcgagt gcaccatctc caaagccaaa gggcagtgcc gagaaccaca ggtgtacacc 360

ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 420

ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 480

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 540

accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 600

tgtctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa a 651

Claims (7)

1. a kind of Fc segments of saltant type humanized IgG, it is characterised in that：The Fc segments of the saltant type humanized IgG be by It is obtained after introducing disulfide bond in the CH2 structural domains of the Fc segments of humanized IgG and CH3 structural domains.

2. the Fc segments of saltant type humanized IgG according to claim 1, it is characterised in that：The Fc of the saltant type humanized IgG The amino acid of the 242nd, the 334th, the 343rd and the 431st is cysteine in the amino acid sequence of segment.

3. the Fc segments of saltant type humanized IgG according to claim 2, it is characterised in that：The Fc of the saltant type humanized IgG The amino acid full length sequence of segment is Seq ID No.1.

4. the Fc segments of saltant type humanized IgG according to claim 3, it is characterised in that：The Fc of the saltant type humanized IgG The coding gene sequence of segment is Seq ID No.2.

5. a kind of preparation method of the Fc segments of saltant type humanized IgG described in claim 2, its step are as follows：

(1) gene of the Fc segments of humanized IgG is obtained；

(2) design primer is amplified and is mutated by the designated amino acid base mutation in Fc into the base of encoding aminothiopropionic acid Type Fc；

(3) carrier for expression of eukaryon is built with saltant type Fc gene orders, transfection mammalian cell carries out eukaryotic expression, purifying Obtain target Fc segments.

6. application of the Fc segments of saltant type humanized IgG described in claim 1 in Fc fusion proteins are prepared.

7. application of the Fc segments of saltant type humanized IgG described in claim 1 in monoclonal antibody is prepared.