CN110305874A - Gerbil immunoglobulin IgG1, IgG2 recombinant protein, gene and application thereof - Google Patents

Abstract

The invention discloses gerbil immunoglobulin IgG1, IgG2 recombinant protein, gene and application thereof. Clawed gerbil immunoglobulin IgG1 recombinant protein, including Clawed gerbil IgG1 heavy chain constant region CH2, CH3, and N-terminal histidine tag, the amino acid sequence is shown in SEQ ID No.3, nucleotide The sequence is shown as SEQ ID No.2. Clawed gerbil immunoglobulin IgG2 recombinant protein, including Clawed gerbil IgG2 heavy chain constant region CH2, CH3, and N-terminal histidine tag, the amino acid sequence is shown in SEQ ID No.4, nucleotide The sequence is shown as SEQ ID No.1. Recombinant proteins are used as immune antigens for antibody preparation, or for immune detection. Compared with conventionally isolated and purified IgG from gerbil serum, the recombinant protein provided by the present invention has the characteristics of strong specificity, good repeatability, high purity and the like.

Description

Gerbil immunoglobulin IgG1, IgG2 recombinant protein, gene and application thereof

technical field

The invention belongs to the field of biotechnology, and in particular relates to the preparation and application of a recombinant protein of heavy chain constant regions CH2 and CH3 of long-clawed gerbil immunoglobulin IgG1 and IgG2.

Background technique

The gerbil (Meriones unguiculatus, Mongolian gerbil) is between the size of rats and mice. It has been used as an experimental animal in the medical field for a long time. It is susceptible to many pathogens. Compared with other rodent experimental animals The long-clawed gerbil can show pathological characteristics and clinical symptoms more similar to humans after being infected with certain pathogens. Therefore, in recent years, the long-clawed gerbil has been increasingly used in various microorganisms (bacteria/virus) and Research fields such as parasitic infection. However, there are still many things to be perfected for the establishment of immunological indicators of gerbils after pathogen infection, especially the establishment of specific antibody serological detection system. In other words, the detection of specific antibodies/immunoglobulins (Immunoglobulin, Ig) such as IgG or IgM induced by exogenous pathogens after infection of gerbils still lacks corresponding specific secondary antibodies, namely anti-antibodies. Research on the immune response of the gerbil, especially the humoral immune response, has been largely limited.

The detection of antibody production in gerbils was initially (in 2002) using indirect agglutination reaction and enzyme-linked immunosorbent assay (ELISA) using anti-mouse IgG polyclonal antibody, which may affect the accuracy and specificity of the experimental results sex. On the other hand, in recent years, different domestic research groups have used Protein G affinity chromatography to purify IgG in gerbil serum, and then used it as an immunogen to prepare rabbit antiserum/polyclonal antibody. The content of IgM in the serum of gerbils is higher than that of other animals, but the content of IgG is not high. Since Protein G can also adsorb other antibody molecules such as IgM in the gerbil serum, the gerbil IgG they purified is likely to contain other antibody molecules such as IgM. Therefore, the rabbit anti-gerbil IgG polyclonal antibody prepared by them is also likely to cross-recognize other antibody molecules of gerbil such as IgM, which will cause deviation in the results of the next step such as ELISA.

Ukaji et al. determined the partial gene sequence encoding 291 amino acids of the heavy chain constant region of the long-clawed gerbil IgG1 and 251 amino acids of the constant region of the IgG2 heavy chain [Ukaji T, Exp Anim, 2012, 61(2):99-107; Ukaji T , Anim SciJ, 2011, 82(5):713-716.]. The protein/amino acid sequence comparison analysis of murine IgG heavy chain constant region showed that the identity between the gerbil IgG1 heavy chain constant region and IgG2 heavy chain constant region was 72.4%, and the identity of the former with the mouse IgG1 heavy chain constant region The highest is 78.0%, and the latter has the highest identity of 84.6% with the IgG heavy chain constant region of hamster. Although the heavy chain constant regions of gerbil IgG1 and IgG2 are 67.0%-78.0% and 78.1%-81.8% identical to mouse IgG1, IgG2a, IgG2b, and IgG3 heavy chain constant regions, respectively, the commercially available rabbit or Goat anti-mouse IgG polyclonal antibody cannot cross-recognize gerbil IgG [Zhang Feng, Chinese Journal of Comparative Medicine, 2017, 27(1):37-42].

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides preparation and application of a recombinant protein of heavy chain constant regions CH2 and CH3 of long-clawed gerbil IgG1 and IgG2.

A gene of recombinant protein of long-clawed gerbil IgG1 immunoglobulin, including CH2, CH3 of constant region of long-clawed immunoglobulin IgG1 heavy chain, and a histidine tag at N-terminal, the nucleotide sequence is as SEQ ID No.1 shown.

A gerbil immunoglobulin IgG1 recombinant protein, including CH2, CH3, and N-terminal histidine tag of the heavy chain constant region of the gerbil IgG1 immunoglobulin, the amino acid sequence of which is shown in SEQ ID No.3.

A gene of recombinant protein of long-clawed gerbil IgG2 immunoglobulin, including CH2, CH3 of heavy chain constant region of long-clawed immunoglobulin IgG2, and a histidine tag at N-terminal, the nucleotide sequence is as SEQ ID No.2 shown.

A gerbil immunoglobulin IgG2 recombinant protein, including CH2, CH3 of the heavy chain constant region of the gerbil IgG2 IgG2, and a histidine tag at the N-terminus, the amino acid sequence of which is shown in SEQ ID No.4.

A set of genes for recombinant proteins of long-clawed gerbil IgG1 and IgG2, including: CH2, CH3, and N-terminal histidine tags of the heavy chain constant region of long-clawed gerbil IgG1 IgG, the nucleotide sequence of which is shown in SEQ ID Shown in No.1: CH2, CH3, and N-terminal histidine tag of the constant region of the IgG2 heavy chain of long-clawed gerbil IgG2, the nucleotide sequence is shown in SEQ ID No.2.

A group of long-clawed gerbil immunoglobulin IgG1 and IgG2 recombinant proteins, including: long-clawed gerbil IgG1 heavy chain constant region CH2, CH3, and N-terminal histidine tag, the amino acid sequence is as shown in SEQ ID No.3 Shown; the CH2, CH3, and N-terminal histidine tag of the heavy chain constant region of the long-clawed IgG2 immunoglobulin of gerbil, the amino acid sequence is shown in SEQ ID No.4.

The application of the recombinant protein is used as an immune antigen for antibody preparation, or for immune detection.

The application of the gene of the recombinant protein is cloned into the expression plasmid pET-28a(+).

Beneficial effects of the present invention:

The recombinant proteins of CH2 and CH3 in the heavy chain constant regions of gerbil IgG1 and IgG2 were respectively used as immunogens to prepare specific polyclonal antibodies, which can be used as secondary antibodies for enzyme-linked immunosorbent assays and other purposes. It provides an effective tool for the serological detection of the gerbil-specific antibody, and is helpful for the standardization and popularization of the gerbil as an experimental animal.

Description of drawings

Figure 1 is a schematic diagram of recombinant expression plasmid encoding;

Fig. 2 is the expression of gerbil IgG1CH23 recombinant protein and purified product electrophoresis and Western blotting figure;

Fig. 3 is the expression and purification product electrophoresis picture and Western blotting picture of gerbil IgG2CH23 recombinant protein;

Fig. 4 is the electrophoresis diagram of rabbit anti-gerbil IgG1CH23 and IgG2CH23 polyclonal antibody;

Figure 5 is the rabbit anti-gerbil IgG1CH23 and IgG2CH23 polyclonal antibodies used in western blot immunotechnique (WB);

Figure 6 shows the rabbit anti-gerbil IgG1CH23 and IgG2CH23 polyclonal antibodies used in enzyme-linked immunosorbent assay (ELISA). In the figure, 1+2: primary antibody is gerbil anti-CVA16 serum, secondary antibody is HPP-labeled rabbit anti-gerbil IgG1CH23 and IgG2CH23 polyclonal antibody; 1: primary antibody is gerbil anti-CVA16 serum, secondary antibody is HPP-labeled rabbit anti- Gerbil IgG1CH23 polyclonal antibody; 2: primary antibody is gerbil anti-CVA16 serum, secondary antibody is HPP-labeled rabbit anti-gerbil IgG2CH23 polyclonal antibody; C: primary antibody is gerbil non-immune serum control, secondary antibody is HPP-labeled rabbit Anti-gerbil IgG1CH23 and IgG2CH23 polyclonal antibodies.

Detailed ways

The present invention will be further elaborated below in conjunction with the embodiments and the accompanying drawings.

Recombinant protein, including gerbil IgG1 heavy chain constant region CH2, CH3 or IgG2 heavy chain constant region CH2, CH3. The above recombinant protein is based on the reported CH2 and CH3 segments of the long-clawed gerbil IgG1 heavy chain constant region (GenBank sequence number AB663132) and IgG2 heavy chain constant region (GenBank sequence number AB597231). In order to facilitate the high-efficiency expression of the recombinant protein in E. coli, the corresponding gene sequence was codon-optimized and fused with a histidine (His) tag at the N-terminus, and cloned into the expression plasmid pET-28a(+) to obtain the recombinant plasmid pET -28a(+)-gerbil IgG1CH23 and pET-28a(+)-gerbil IgG2-CH23 were transformed into Escherichia coli BL21(DE3) competent cells, and the transformants were screened to obtain recombinant expression bacteria. After the recombinant protein was induced to express and purified, it was used as an immunogen to inoculate rabbits, and after obtaining antiserum, it was purified by affinity antibody chromatography to obtain specific polyclonal antibodies.

For the convenience of recombinant protein purification, affinity purification tags can be used. The recombinant protein includes a histidine (His) tag sequence.

The amino acid sequence of the recombinant protein, CH2 and CH3 of the heavy chain constant region of the gerbil IgG1 IgG1, is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.1.

The amino acid sequence of CH2 and CH3 of the heavy chain constant region of the gerbil IgG2 IgG2 is shown in SEQ ID No.4, and the nucleotide sequence is shown in SEQ ID No.2.

Example 1 Construction and transformation of recombinant plasmids pET-28a(+)-gerbil IgG1CH23 and pET-28a(+)-gerbil IgG2-CH23

According to the protein sequences of the heavy chain constant regions of the long-clawed gerbil immunoglobulin IgG1 and IgG2, select their respective constant domains CH2 and CH3, artificially synthesize DNA fragments after codon optimization, and introduce corresponding restriction endonucleases at both ends The NcoI and XhoI sites were digested with enzymes and cloned into the E. coli expression plasmid pET-28a(+), so that the N-terminus of the target protein was tagged with His to facilitate purification, and the corresponding recombinant plasmid was identified correctly. The schematic diagram of recombinant expression plasmid encoding is shown in Figure 1.

The recombinant plasmid pET-28a(+)-IgG1CH23 expresses the constant domains CH2 and CH3 of gerbil IgG1, encoding 218 amino acids and a theoretical molecular weight of 24.8 kDa; the recombinant plasmid pET-28a(+)-IgG2CH23 expresses the constant domains CH2 and CH3 of gerbil IgG2 CH3, encoding 187 amino acids, has a theoretical molecular weight of 21.2 kDa.

The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and the transformants were screened to obtain recombinant expressing bacteria.

Example 2 Expression and purification of recombinant proteins of long-clawed gerbil IgG1-CH23 and IgG2-CH23

Recombinant plasmids pET-28a(+)-IgG1CH23 and pET-28a(+)-IgG2-CH23 were transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB plates and cultured at 37°C overnight, and single colonies were picked and inoculated in LB In liquid medium, cultivate until the absorbance A ₆₀₀ reaches 0.8, then add IPTG (1 mmol/L) to induce overnight at 25°C.

The next day, Escherichia coli bacteria were collected by centrifugation, resuspended in buffer, and then ultrasonically crushed. The supernatant and precipitate were collected by centrifugation, and the expression form of the recombinant protein was analyzed by sodium dodecylsulfonate polyacrylamide gel electrophoresis (SDS-PAGE). Dissolve the broken bacteria pellet in 8 mol/L urea solution, collect the supernatant after centrifugation, pass through the His GravitrapTM column under denaturing conditions, use the imidazole concentration gradient to elute and collect the target protein, and then in the dialysate (10 mmol/L Tris , 20 mmol/L NaCl [pH9.0]) at 4°C for renaturation and gradual removal of urea.

The expression and purification of the gerbil IgG1CH23 recombinant protein are shown in Figure 2 by electrophoresis and Western blot.

Figure 3 shows the electrophoresis and Western blotting of the expression and purification product of the gerbil IgG2CH23 recombinant protein.

After the recombinant plasmids were transformed into Escherichia coli, the target protein could be highly expressed after being induced by IPTG, and all of them existed in the form of inclusion body and the molecular weight was in line with the expectation. A His tag was introduced into the N-terminus of both to facilitate purification, so WB showed specific positive reaction with anti-His antibody.

Example 3 Preparation of Rabbit Anti-Clawed Gerbil IgG1-CH23 and IgG2-CH23 Polyclonal Antibodies

The above-mentioned purified recombinant protein gerbil IgG1 CH23 and IgG2 CH23 were inoculated into New Zealand big-eared white rabbits respectively as immunogens, injected subcutaneously at multiple points, blood was collected four weeks after the initial immunization, serum was separated and used for Hitrap Protein A affinity antibody chromatography column Polyclonal antibodies were obtained by purification.

The electropherogram of the rabbit anti-gerbil IgG1CH23 or IgG2CH23 polyclonal antibody is shown in Figure 4.

Example 4 Rabbit Anti-Clawed Gerbil IgG1 CH23 and IgG2 CH23 Polyclonal Antibody Used in Western Blot Immunotechnique (WB)

After the above rabbit anti-gerbil IgG1 CH23 and IgG2 CH23 polyclonal antibodies were labeled with horseradish peroxidase (HRP), the recombinant protein expressed in Escherichia coli can be specifically recognized by Western blot (WB) Gerbil IgG1 CH23 and IgG2 CH23 (Figure 5).

Example 5 Rabbit anti-gerbil IgG1 CH23 and IgG2 CH23 polyclonal antibodies used in enzyme-linked immunosorbent assay (ELISA)

The inactivated Coxsackie virus A16 type YY157 strain was used as the capture antigen to coat the plate, the gerbil anti-CVA16 serum or the control non-immune serum was used as the primary antibody, and the HRP-labeled rabbit anti-gerbil IgG1 CH23 and IgG2 CH23 The polyclonal antibody was used as a secondary antibody/anti-IgG antibody in ELISA to detect the anti-CAV16-specific IgG antibody induced by Coxsackie virus A16 (CAV16) infection in gerbils (Figure 6).

The results of the above examples show that the recombinant proteins of IgG1 CH2 and CH3 of long-clawed gerbils and the recombinant proteins of IgG2 heavy chain constant domain CH2 and CH3 were respectively expressed by means of molecular cloning, and specific rabbit polyclonal antibodies were successfully prepared as immunogens. The serological detection of gerbil-specific antibodies provides an effective tool and helps standardize and promote the use of gerbils as experimental animals.

sequence listing

<110> Zhejiang Cancer Hospital

<120> Gerbil immunoglobulin IgG1, IgG2 recombinant protein, gene and application thereof

<141> 2019-06-19

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 665

<212>DNA

<213> Artificial Sequence

<400> 1

ccatgggtca tcaccaccac catcacggcc tggttccgcg tggcagcgtg ccggaagttt 60

cttccgtgtt catctttcca ccgaaaccga aagatgttct gactatctcc ctgactccga 120

agatcacctg cgtggtggtt gacatcagcc aggacgatcc agaggtggag atcagctggt 180

tcgttgacga tgtggaagta cacactgccc agacccagcc acgtgaagaa cagtttaact 240

ctactttccg tgtggtatcc accctgccga tcctgcacca ggactggctg aatggtaaag 300

agttcaaatg caaagtgaac agcgaagcat ttccgagccc aatcgagaag accgtgagca 360

agatgaaagg tcagccgcag gcaccgcagg tttacatgat gccaccaccg aaagagcaga 420

tgactaagaa cgaggtaact gtttcctgct acgttggcgg tttctatccg ccagatgttt 480

ccgtagagtg gcagcgcaat ggcgagccgg aaccgaactt caaatctacc cagccgaccc 540

tggacactga cgagtcctac ttcctgtact ctaaactgga tgtaaagcgt gcaaactggg 600

agaaaggtga cgtgttcact tgctccgtgc tgcacgaggc actgcacaat catcactaac 660

tcgag 665

<210> 2

<211> 572

<212>DNA

<213> Artificial Sequence

<400> 2

ccatgggcca tcaccaccac catcacggtc tggttccgcg tggcagcgct ccagacctgt 60

ctggcggtcc gtccgtgttt atcttcccac caaagccgaa agatgtgctg atgatgaccc 120

tgactccgaa gatcacctgc gtggtagtag acgtttccga ggacgaaccg gatgttcagt 180

tcaactggtt cgttgacaat gtggaagtgc ataacgcgca gacccaaccg cgtgagggtc 240

agtacaactc taccttccgt gtagtgtctg cgctgccgat taagcaccaa gactggatgt 300

ctggcaaaga gttcaaatgc aaagttaaca acaaagcact gccgtctccg atcgagaaga 360

ccatttccaa gccaaagggt ccggttcgtg agccgcaggt ttacgccttt ccaccaccga 420

aggaacagat gaaccgtaac cagctgtctc tgacctgcat gattaccaac ttctatccgg 480

aagcgatcga cgtggaatgg gaacgcaacg gccaggaaga gcgtaactac aagaacacca 540

aaccagtgct ggattccgat ggctaactcg ag 572

<210> 3

<211> 218

<212> PRT

<213> Artificial Sequence

<400> 3

Met Gly His His His His His His His Gly Leu Val Pro Arg Gly Ser Val

1 5 10 15

Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val

20 25 30

Leu Thr Ile Ser Leu Thr Pro Lys Ile Thr Cys Val Val Val Asp Ile

35 40 45

Ser Gln Asp Asp Pro Glu Val Glu Ile Ser Trp Phe Val Asp Asp Val

50 55 60

Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Phe Arg Val Val Ser Thr Leu Pro Ile Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Phe Lys Cys Lys Val Asn Ser Glu Ala Phe Pro Ser

100 105 110

Pro Ile Glu Lys Thr Val Ser Lys Met Lys Gly Gln Pro Gln Ala Pro

115 120 125

Gln Val Tyr Met Met Pro Pro Pro Lys Glu Gln Met Thr Lys Asn Glu

130 135 140

Val Thr Val Ser Cys Tyr Val Gly Gly Phe Tyr Pro Pro Asp Val Ser

145 150 155 160

Val Glu Trp Gln Arg Asn Gly Glu Pro Glu Pro Asn Phe Lys Ser Thr

165 170 175

Gln Pro Thr Leu Asp Thr Asp Glu Ser Tyr Phe Leu Tyr Ser Lys Leu

180 185 190

Asp Val Lys Arg Ala Asn Trp Glu Lys Gly Asp Val Phe Thr Cys Ser

195 200 205

Val Leu His Glu Ala Leu His Asn His His

210 215

<210> 4

<211> 187

<212> PRT

<213> Artificial Sequence

<400> 4

Met Gly His His His His His His His Gly Leu Val Pro Arg Gly Ser Ala

1 5 10 15

Pro Asp Leu Ser Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro

20 25 30

Lys Asp Val Leu Met Met Thr Leu Thr Pro Lys Ile Thr Cys Val Val

35 40 45

Val Asp Val Ser Glu Asp Glu Pro Asp Val Gln Phe Asn Trp Phe Val

50 55 60

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

65 70 75 80

Tyr Asn Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Lys His Gln

85 90 95

Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala

100 105 110

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

115 120 125

Arg Glu Pro Gln Val Tyr Ala Phe Pro Pro Pro Lys Glu Gln Met Asn

130 135 140

Arg Asn Gln Leu Ser Leu Thr Cys Met Ile Thr Asn Phe Tyr Pro Glu

145 150 155 160

Ala Ile Asp Val Glu Trp Glu Arg Asn Gly Gln Glu Glu Arg Asn Tyr

165 170 175

Lys Asn Thr Lys Pro Val Leu Asp Ser Asp Gly

180 185

Claims (8)

1. A gene of a long-clawed gerbil immunoglobulin IgG1 recombinant protein, characterized in that it comprises a long-clawed gerbil immunoglobulin IgG1 heavy chain constant region CH2, CH3, and a histidine tag at the N end, nucleotide sequence As shown in SEQ ID No.1. 2. A long-clawed gerbil immunoglobulin IgG1 recombinant protein, characterized in that it comprises the constant region CH2, CH3 of the long-clawed gerbil IgG1 heavy chain, and a histidine tag at the N end, and the amino acid sequence is as SEQ ID No .3 shown. 3. A gene of a long-clawed gerbil immunoglobulin IgG2 recombinant protein, characterized in that it comprises a long-clawed gerbil immunoglobulin IgG2 heavy chain constant region CH2, CH3, and a histidine tag at the N-terminal, nucleotide sequence As shown in SEQ ID No.2. 4. A long-clawed gerbil immunoglobulin IgG2 recombinant protein, characterized in that it comprises the constant region CH2, CH3 of the long-clawed gerbil IgG2 heavy chain, and a histidine tag at the N-terminal, and the amino acid sequence is as SEQ ID No .4 shown. 5. A group of genes of long-clawed gerbil immunoglobulin IgG1 and IgG2 recombinant protein, characterized in that, comprising: CH2, CH3 of the constant region of long-clawed gerbil IgG1 heavy chain, and a histidine tag at the N-terminal, nuclear The nucleotide sequence is shown in SEQ ID No.1; the gerbil IgG2 heavy chain constant region CH2, CH3, and the N-terminal histidine tag, the nucleotide sequence is shown in SEQ ID No.2. 6. A group of long-clawed gerbil immunoglobulin IgG1 and IgG2 recombinant proteins, characterized in that, comprising: long-clawed gerbil immunoglobulin IgG1 heavy chain constant region CH2, CH3, and N-terminal histidine tag, the amino acid sequence is as follows Shown in SEQ ID No.3; CH2, CH3, and N-terminal histidine tag of the constant region of the IgG2 heavy chain of long-clawed gerbil immunoglobulin, and the amino acid sequence is shown in SEQ ID No.4. 7. The recombinant protein application according to any one of claims 2, 4, and 6, characterized in that it is used as an immune antigen for antibody preparation, or for immune detection. 8. The genetic application of the recombinant protein according to any one of claims 1, 3, and 5, characterized in that it is cloned into the expression plasmid pET-28a(+).