CN108103068B - Method for preparing nano antibody of anti-carcinoembryonic antigen - Google Patents

Abstract

The invention provides a method for preparing a nano antibody against carcinoembryonic antigen, which comprises the following steps: (1) amplifying the coding gene of the carcinoembryonic antigen nano antibody; (2) connecting the coding gene obtained in the step (1) to an expression vector pKLAC 2; (3) transferring the vector obtained in the step (2) into a host cell Kluyveromyces lactis; (4) inducing the host cell obtained in the step (3) and harvesting the expressed nano antibody against the carcinoembryonic antigen. The invention also provides an expression vector containing the carcinoembryonic antigen nano-antibody and a host cell Kluyveromyces lactis containing the vector. By adopting the method, the expressed nano antibody is secreted out of the cell, the foreign protein is less, and the target band for Western Blot detection is single, so that the cost of separation and purification is reduced, and the expression efficiency is greatly improved; wherein the extracellular secretion amount is 0.8mg/l under the condition of shake flask culture, and the nano antibody affinity of the carcinoembryonic antigen is 5.042 x 10 measured by a direct coupling method^‑9。

Description

Method for preparing nano antibody of anti-carcinoembryonic antigen

Technical Field

The invention relates to a preparation method of an antibody protein, and more particularly relates to a method for preparing a nano antibody protein by using kluyveromyces lactis.

Background

Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a glycoprotein with a molecular weight of about 180 kDa. CEA is a member of the immunoglobulin superfamily, originally classified as a protein expressed only in fetal tissues, and has now been identified in normal adult tissues. Overexpression of CEA is detected in colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers. Therefore, CEA has been identified as a tumor associated antigen and also has been used as a tumor marker, and immunological assays for measuring elevated CEA in the blood of cancer patients have been used clinically for the prognosis and control of cancer.

Currently, CEA has become a potential tumor-associated antigen for targeted therapy. CEA-targeted immunotherapy of cancer has 2 approaches, one of which is the use of anti-CEA antibodies to elicit the lytic activity of immune cells, particularly by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), to eliminate CEA-expressing tumor cells; another way is to specifically target CEA-expressing tumor cells by conjugating the anti-CEA antibody or antibody fragment with effector molecules such as drugs, toxins, radionucleotides, immunomodulators or cytokines, thereby exerting the therapeutic effect of the effector molecules.

1993 Belgian scientists found that in alpaca peripheral blood, there exists a naturally light chain-deficient antibody comprising only one heavy chain variable region and two conventional CH₂And CH₃And (4) a zone. Since the antibody is a variable region sequence after the constant region is removed, the molecular weight of the antibody is about 15kDa and the diameter of the antibody is 10 nanometers, and the antibody is also called a nanobody. Furthermore, such single domain antibodies are also observed in sharks. Compared with the traditional four-chain antibody, the nano-antibody has the affinity equivalent to that of the traditional antibody, but is superior to the traditional antibody in the aspects of solubility, stability, resistance to aggregation, refolding property, expression yield, DNA operation and the like.

Considering the simplicity and high efficiency of industrial application, the expression method of the nanobody in escherichia coli is mostly adopted at present, but the disadvantage of the expression method is that the antibody cannot be modified by glycosylation, and then the nanobody contains endotoxin, and the disadvantages limit the application of the nanobody in restriction chemistry and clinical medicine. Although pichia pastoris is used as an expression host bacterium, the pichia pastoris can also successfully express and secrete the nano antibody, methanol is required to be used as an inducer in the expression process of the nano antibody, so certain disadvantages are brought: firstly, excessive methanol is added to generate certain toxicity on yeast, so that the yield of the nano antibody is influenced; secondly, the methanol per se does not accord with the safety level of food and medicine, and the methanol needs to be further removed subsequently, so the operation is more complicated. The saccharomyces cerevisiae expression system has the defects of low fermentation density, poor secretion expression capability, excessive glycosylation and the like. In consideration of various aspects, the kluyveromyces lactis is the best choice in yeast expression systems, such as whether the product can be secreted to the extracellular space, the stability of the product, whether special byproducts can be generated, the safety level of food and medicines, and the like. At present, some medicinal proteins are successfully expressed and applied.

Kluyveromyces lactis is one of the very important yeasts in the field of biotechnology science, and has important significance in the food safety application industry and the industrial large-scale production of enzymes. Kluyveromyces lactis has been used in the food industry as a protein expression system. Due to the unique characteristics of the nano antibody in the aspects of molecular weight, spatial structure and the like, compared with the traditional Kluyveromyces lactis expression, the nano antibody for expressing the carcinoembryonic antigen (Anti-CEA) in yeast cells has not been reported at present. However, considering that the kluyveromyces lactis is used as an engineering bacterium, the most important point is that the kluyveromyces lactis is edible yeast which can reach the food safety level and is determined by the U.S. Food and Drug Administration (FDA) and the ministry of health in china, and the advantage of eliminating special byproducts is not required.

Disclosure of Invention

Based on the above objects, the present invention provides, in a first aspect, a method for preparing a nanobody against carcinoembryonic antigen (CEA), comprising the steps of:

(1) amplifying the coding gene of the carcinoembryonic antigen nano antibody;

(2) connecting the coding gene obtained in the step (1) to an expression vector pKLAC 2;

(3) transferring the vector obtained in the step (2) into a host cell Kluyveromyces lactis;

(4) inducing the host cell obtained in the step (3) and harvesting the expressed nano antibody against the carcinoembryonic antigen.

In a preferred embodiment, the coding gene in step (1) is as shown in SEQ ID NO.1, and the coding gene is a nucleotide sequence optimized for expression in a host cell, i.e., Kluyveromyces lactis, without changing the amino acid sequence of the anti-carcinoembryonic antigen nanobody.

In a more preferred embodiment, the ligation described in step (2) is the insertion of the coding gene between a NotI site starting at base 304 and an EcoRI site starting at position 329 of the expression vector pKLAC 2.

In a particularly preferred embodiment, the host cell of step (3) is Kluyveromyces lactis GG 799.

In a most preferred embodiment, the inducing in step (4) is performed for 72 hours under the growth condition that the pH value is 7.2 and the carbon source of the culture medium is lactose.

Secondly, the invention also provides an expression vector pKLAC2 containing the carcinoembryonic antigen nano-antibody.

In a preferred embodiment, the encoding gene of the carrier anti-carcinoembryonic antigen nanobody is shown in SEQ ID NO. 1.

In a more preferred embodiment, the coding gene is inserted between the NotI site starting at base 304 and the EcoRI site starting at position 329 of the expression vector pKLAC2 and linked to the expression vector.

Finally, the invention also provides a host cell containing any one of the expression vectors, wherein the host cell is Kluyveromyces lactis.

In a preferred embodiment, the kluyveromyces lactis is strain GG 799.

The invention inserts the coding gene of the CEA antigen-resistant nano antibody 11C12 into an expression vector pKLAC2 to construct pKLAC2-11C 12. The coding gene of the nano antibody is optimized under the condition of not changing an amino acid sequence, the expression plasmid is introduced into Kluyveromyces lactis GG799, the culture condition is optimized, the culture temperature is 30 ℃, the initial pH is 7.2, the rpm is 200, and the nano antibody for expressing the CEA antigen is induced by galactose after being cultured for 24 hours. By adopting the method of the invention, the cell density reaches OD after shaking culture for 72 hours₆₀₀30, the extracellular secretion protein can reach 0.8mg/l, and the molecular size is 12.5 KDa. The method provided by the invention is simple and efficient, reduces the purification cost, and lays a foundation for the industrial preparation of the carcinoembryonic antigen-resistant nano antibody.

Drawings

FIG. 1 shows a flow chart of construction of expression plasmid pKLAC2-11C 12;

FIG. 2 is an electrophoretogram of an amplification product of a target gene;

FIG. 3 shows the electrophoresis chart of the restriction enzyme recovery of the target gene;

FIG. 4 is a PCR identification chart of a target gene of a recombinant bacterium;

FIG. 5 shows a counterstained electrophoretogram and a WB diagram of GG 799-expressing protein of Kluyveromyces lactis;

FIG. 6 is a graph of affinity assay for Kluyveromyces lactis GG 799-expressed protein.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

The percentage contents referred to in the following examples are all mass percentages, and the conventional experimental operation steps such as enzyme digestion, ligation, recovery, transformation, PCR amplification and the like are described in molecular cloning (third edition). Primer synthesis and sequencing were performed by Qingdao Okagaku Biopsis.

Example 1 preparation of Nanobodies against carcinoembryonic antigen

1. Nano antibody gene clone of anti carcinoembryonic antigen

1.1 primer design

According to the sequence which is obtained by screening in the early stage and can detect the CEA gene, the nucleotide sequence is optimized (SEQ ID NO.1) on the premise of not changing the amino acid sequence (SEQ ID NO.2), and then the following pair of primers are designed and synthesized according to the characteristics of the polyclonal site of the Kluyveromyces lactis expression vector pKLAC 2:

the pKL1 forward primer was:

5’-ccatggGCGGCCGCGAATCTGGTGGTGGTTTGGTTCAA-3’；

the pKL2 reverse primer was:

5’-AGGgaattcTCAATGATGATGATGATGATGTTGAGTACCTTGACCGA AGTAACC-3’

NotI and EcoRI cleavage sites are designed at both ends of pKL1 and pKL2 (see the lower case and underlined part of the above sequence)

1.2 PCR amplification of Anti-CEA Gene 11C12

PCR is carried out by adopting pKL1 and pKL2 primers and using a pPICZ alpha A-11C12 vector constructed in a laboratory as a template, wherein the reaction system is as follows:

the total volume is 50ul, and the reaction conditions are as follows: 2 minutes at 98 ℃; 30 seconds at 98 ℃, 30 seconds at 55 ℃ and 1 minute at 72 ℃ for 30 times of circulation; 10 minutes at 72 ℃.

1.3 recovery of the fragment of interest from the PCR reaction product

A target gene fragment is purified and recovered from a PCR product by adopting a gel cutting and column passing method, the PCR reaction product is subjected to agarose gel electrophoresis, and then a target gene 11C12 (figure 2, the length of the target gene is 315bp, wherein M1 is DNA standard molecular weight (Kb), corresponding bands from top to bottom are respectively 2000, 1000, 750, 500, 250 and 100, M2 is DNA standard molecular weight (Kb), corresponding bands from top to bottom are respectively 12000, 8000, 6000, 5000, 4000, 3000, 2500, 2000, 1500, 1000 and 500, a Lane 1 is a target gene DNA enzyme digestion recovery fragment, a figure 3 is a pKLAC2 carrier double enzyme digestion recovery fragment) is cut and recovered according to a DNA recovery kit specification (purchased from Beijing Tiangen biotechnology Limited and product number is DP 209-02).

1.4 ligation of the Gene of interest to the Kluyveromyces lactis expression vector pKLAC2 (purchased from NEW ENGLAND BioLabs, product No. # N3742S)

The recovered target gene 11C12 and pKLAC2 vectors are subjected to double enzyme digestion by using restriction enzymes NotI and EcoRI respectively, then a target fragment is recovered, and T is used₄The plasmid pKLAC2-11C12 was obtained by ligating the above-mentioned fragments with ligase, and the construction scheme is shown in FIG. 1. The constructed pKLAC2-11C12 plasmid was transformed into DH5 alpha (product No. # CW080 0808B, available from Beijing Kangji reagent Biotech Co., Ltd.).

2. Transformation and expression

2.1 preparation of Kluyveromyces lactis GG799 (purchased from NEW ENGLAND BioLabs, product number # C1001S) electroporation competent cells and electroporation thereof

(1) GG799 single colonies activated on a YPD solid medium were inoculated into 10ml of a fresh YPD liquid medium, and cultured overnight at 30 ℃ with shaking at 250 rpm;

(2) the overnight culture was inoculated into 100ml of fresh YPD liquid medium to obtain the initial concentration OD₆₀₀0.2. Culturing for 3h to make yeast cell enter early stage of log phase when OD is₆₀₀0.8-1.4 (1 × 10)⁷Per ml), centrifuging at 4 ℃ and 3000rpm for 5 minutes, washing the cells once with sterile deionized water, and centrifuging to collect thalli;

(3) the collected cells were suspended in 20ml of incubation buffer to a cell density of about 2X 10⁸And/ml. Incubating at 30 ℃ and 100rpm for 30 minutes;

(4) collecting thallus at 4 deg.C and 3000rpm for 5 min, mixing thallus with 1ml precooled EB buffer solution, and controlling low temperature in ice box;

(5) subpackaging the mixed solution with 100ul per tube;

(6) 100ul of competent cells were mixed with 1ug of the sample of pKLAC2-11C12 to be transformed. Ice-bath and standing for 15 minutes;

(7) adding the mixture into a pre-cooled 0.2cm electric rotating cup;

(8) setting the condition of electric conversion to be 1.5 kV; a capacitance of 25 μ F; the resistance is 200 omega. The electric shock is carried out once, and the electric shock time is ensured to be within the range of 4-10 msec;

(9) immediately adding 1.0mL of precooled 1M sorbitol after the electric shock is finished, gently mixing uniformly, transferring into a 1.5mL EP tube, standing for 1h at 30 ℃, centrifuging for 3 minutes at 4000rpm, and discarding about 100ul of sorbitol from 95% of supernatant;

(10) the bacterial solution was applied to yeast minimal carbon source medium YCB (100mL) containing 5mM acetamide: 3ml of 1M Tris-HCl, 1.17g of YCB, 2g of agarose, 0.03g of acetamide, cultured at 30 ℃ for 3-5 days, and observed.

2.2 screening and identification of Kluyveromyces lactis Positive transformants

2.2.1 screening of Kluyveromyces lactis Positive transformants

The transformed cell mixture was plated on YCB (formula 2.1) which is the basic carbon source medium for yeast containing 5mM acetamide. YCB medium contains nutrients and carbon sources required for k.lactis growth, but lacks nitrogen sources. Recombinants can grow into larger colonies as long as the acetamidase expressed by the amds gene on pKLAC2 degrades the acetamide added to the medium to ammonia, which is available to the cells. Larger colonies were selected as positive transformants based on this principle and further identified.

2.2.2 identification of correctly integrated transformants by the Yeast colony PCR method

Selecting positive bacterial colonies on a plate, picking the bacterial colonies for shake culture, collecting bacterial liquid into a 1.5ml EP tube, centrifuging, pouring out supernatant, adding a small amount of quartz sand into bacterial precipitates, grinding by using a manual plastic grinding rod, adding 100ul of distilled water after grinding for multiple times, centrifuging, and taking the supernatant as a PCR template. PCR amplification was carried out using F1(10X) -ACACACGTAAACGCGCTCGGT and F2(10X) -ATCATCCTTGTCAGCGAAAGC as primers. The correct integration of the expression cassette in the transformants can be verified by PCR, if a 2.4kb fragment is amplified (FIG. 4 verifies correct integration of the expression cassette).

And (3) PCR reaction system:

reaction conditions are as follows: 2 minutes at 98 ℃; 30 seconds at 98 ℃, 30 seconds at 55 ℃ and 1 minute at 72 ℃ for 30 times of circulation; 10 minutes at 72 ℃.

And (4) carrying out small-sized shake flask induction culture on the positive clone which is verified to be correct by the PCR, and carrying out subsequent experiments.

2.3 Kluyveromyces lactis expresses the nano-antibody 11C12 against carcinoembryonic antigen.

2.3.1 PCR-positive strains were streaked onto YPD solid (1% yeast extract, 2% peptone, 2% glucose, 2% agar) plates and left at 30 ℃ for 24-30 hours.

2.3.2 Single clones were inoculated into 50ml of YPD liquid (1% yeast extract, 2% peptone, 2% glucose) medium and cultured at 200-.

2.3.3 inoculating 10ml of the bacterial liquid into 100ml of YPG liquid (1% yeast extract, 2% peptone, 2% lactose) medium, inducing expression with galactose as inducer, and inducing at 30 deg.C and 200rpm for 72 hr.

2.4 identification of Kluyveromyces lactis expression anti-carcinoembryonic antigen Nanobody 11C12

Protein expression was characterized by Western blotting (Western Blot).

2.4.1 Collection and processing of protein samples

And centrifuging the fermented bacterial liquid at 8000rpm for 20 minutes, and collecting a supernatant sample. The sample was added to a 3000D ultrafiltration tube (available from Millipore under the trade designation UFC800324) at 7500g, centrifuged for 30 minutes, and after the sample was concentrated 10-fold, the concentrated sample in the ultrafiltration tube was collected, 20uL of the concentrated Protein sample was mixed with 5uL of 5 XProtein buffer Protein loading buffer (available from Shanghai Biotech Ltd under the trade designation B026549), and denatured at 98 ℃ for 5 minutes. After treatment, SDS-PAGE and Western Blot were performed for validation.

2.4.2SDS-PAGE

After SDS-PAGE precast gel (purchased from Nanjing Jinruis Biotechnology Co., Ltd., product number M01215C) is installed, running buffer is added into an electrophoresis tank, a comb is pulled out to remove bubbles, 20ul of sample is taken for sample loading after the sample is cooled to room temperature, the voltage is 150V, the current is 400mA, the power is 120W, and the time is 55 minutes. After electrophoresis, one piece of glue is used for examination and dyeing, and the other piece of glue is used for film transfer.

2.4.3 transfer film

After electrophoresis, carefully taking out the gel, cutting off the redundant concentrated gel part, and then putting the separation gel into the membrane transfer buffer solution for balance and standby. Cutting a PVDF membrane and 6 pieces of filter paper according to the size of the separation gel, soaking the membrane in methanol for 10-15 seconds, immediately transferring the membrane to ultrapure water for rinsing treatment, and then transferring the membrane and the filter paper to a membrane transferring buffer solution for balancing for standby. The following examples are taken as negative electrodes in the film transfer device: firstly, 3 pieces of filter paper are flatly placed, bubbles are removed, then the membrane is placed on the three layers of filter paper, glue is placed on the membrane, the bubbles are removed, and finally 3 layers of filter paper are placed on the membrane to be made into a sandwich shape. The current of the transfer film was set at 200mA for 20 minutes.

2.4.4 sealing

5% blocking solution (BSA 5g, TBST buffer 100ml) was prepared 10 minutes before the completion of membrane transfer, and after the completion of membrane transfer, the membrane was put into the blocking solution and subjected to blocking treatment at 25 ℃ for 1 hour at 80rpm on a horizontal shaker.

2.4.5 Primary antibody incubation

An HRP-labeled Anti-His-tagged murine Monoclonal Antibody (HRP Conjugated Anti His-Tag Mouse Monoclonal Antibody primary Antibody) (purchased from Beijing kang, century Biometrics, Inc., cat # CW0285) was diluted with 1% BSA (TBST) at a ratio of 1:2000, and the membrane was treated in a primary Antibody incubation solution for 2 hours and then washed 3 times with TBST for 10 minutes each.

2.4.6 visualization and protein detection

According to the size of the membrane, a developing solution is prepared according to an enhanced HRP-DAB substrate developing kit (purchased from Tiangen biochemistry Co., Ltd., product number is PA110), the developing solution is uniformly dripped on the PVDF membrane, and the developing treatment is carried out for 5-10 minutes in a dark place. Immediately after the completion of the color development, the developing solution was washed with water to terminate the color development reaction, and the result of the color development was shown in FIG. 5. Lane 1 shows a protein fraction marker, lane 2 shows a counterstaining pattern of the target gene, and lane 3 shows a Western Blot color. It can be seen that the molecular weight of the expressed nano antibody 11C12 protein against carcinoembryonic antigen is 12.5KDa, and the expressed nano antibody and carcinoembryonic antigen have specific immune binding reaction.

Example 2 determination of affinity of Kluyveromyces lactis expression of Nanobody 11C12 protein against carcinoembryonic antigen

Direct coupling (Biacore) was used to test the affinity of nanobodies against carcinoembryonic antigen. As carcinoembryonic Antigen, a Recombinant Carcino embryo Antigen CEA protein (Abcam, cat # ab99235) was used.

Preparing 20ug/ml nanometer antibody of carcinoembryonic antigen by using running buffer solution as an upper machine sample, selecting a glycine hydrochloride system with pH of 1.5, using a template method carried by the instrument, and setting parameters as follows: the sample introduction time is 60 seconds,the flow rate was 30 ul/min, the dissociation time was 600 sec, the regeneration time was 30 sec, the flow rate was 30 ul/min, and the affinity of the nanobody against carcinoembryonic antigen to carcinoembryonic antigen was tested. As shown in FIG. 6, the nano-antibody affinity against carcinoembryonic antigen measured by direct coupling method was 5.042X 10^-9。

Sequence listing

<110> Shenzhen Shang Nanobody technology Limited

<120> a method for preparing nano antibody against carcinoembryonic antigen

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 315

<212> DNA

<213> Lama pacos

<400> 1

gaatctggtg gtggtttggt tcaaccaggt ggttctttga gattgtcctg tgctgcttcc 60

ggtttcacct tctcatctta cactggtaga tgggacagac tggctccagg taaagaaaga 120

gagttggttg ctactatcac ctccactggt ggttccacta actacgctga ttctgtcaag 180

ggtagattca ccatctccag agacaacgcc aagaacacca tctacttgca gatgaccaag 240

ctgaagccag acgacactgc tgtttactac tgtgttgctc acaacggtag aggttacttc 300

ggtcaaggta ctcaa 315

<210> 2

<211> 105

<212> PRT

<213> Lama pacos

<400> 2

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr Gly Arg Trp Asp

20 25 30

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

35 40 45

Thr Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

50 55 60

Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu Gln Met Thr Lys

65 70 75 80

Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys Val Ala His Asn Gly

85 90 95

Arg Gly Tyr Phe Gly Gln Gly Thr Gln

100 105

Claims (9)

1. A method of preparing nanobodies against carcinoembryonic antigen, the method comprising the steps of:

(1) amplifying the coding gene of the carcinoembryonic antigen nano antibody with the sequence as SEQ ID NO. 1;

(2) connecting the coding gene obtained in the step (1) to an expression vector pKLAC 2;

(3) transferring the vector obtained in the step (2) into a host cell Kluyveromyces lactis;

(4) inducing the host cell obtained in the step (3) and harvesting the expressed nano antibody against the carcinoembryonic antigen.

2. The method according to claim 1, wherein the ligation in step (2) is carried out by inserting the coding gene between NotI site starting at base 304 and EcoRI site starting at site 329 of pKLAC2 in the expression vector.

3. The method of claim 2, wherein the host cell of step (3) is Kluyveromyces lactis GG 799.

4. The method according to claim 3, wherein the inducing of step (4) is to induce the expression of the nano-antibody against carcinoembryonic antigen with galactose after 24 hours of culturing under the growth conditions of culturing at 30 ℃ and pH 7.2, and the carbon source of the culture medium is lactose, and to harvest the expressed antibody after 72 hours.

5. An expression vector containing an anti-carcinoembryonic antigen nano antibody, wherein the coding gene of the anti-carcinoembryonic antigen nano antibody is shown in SEQ ID NO. 1.

6. The expression vector of claim 5, wherein the expression vector is pKLAC 2.

7. The expression vector of claim 6, wherein the coding gene is inserted between and linked to the expression vector pKLAC2 at a NotI site starting at base 304 and an EcoRI site starting at site 329.

8. A host cell comprising the expression vector of any one of claims 5 to 7, wherein the host cell is Kluyveromyces lactis.

9. The host cell of claim 8, wherein the Kluyveromyces lactis is strain GG 799.

Images