CN109336964B

CN109336964B - Cat B lymphocyte stimulating factor cDNA and its coding protein, cloning method and use

Info

Publication number: CN109336964B
Application number: CN201811114164.1A
Authority: CN
Inventors: 张嘉鑫; 武小龙; 张双全
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2018-09-25
Filing date: 2018-09-25
Publication date: 2021-11-12
Anticipated expiration: 2038-09-25
Also published as: CN109336964A

Abstract

The invention discloses a cat B lymphocyte stimulating factor cDNA, a coding protein, a cloning method and application thereof. The base sequence of the cat B lymphocyte stimulating factor cDNA is shown as SEQ ID NO.1, and the amino acid sequence of the coded protein is SEQ ID NO. 2. The cDNA of cat B lymphocyte stimulating factor cBAFF cloned from cat for the first time is subjected to fluorescent quantitative PCR analysis, wherein the cBAFF has expression in each detection tissue, the expression level is the highest in spleen, and the expression level is the lowest in small intestine; indicating that BAFF plays an important role in the immune system of cats. The extracellular region recombinant fusion protein csBAFF of the gene has the activity function of cBAFF, the protein has obvious survival/proliferation promoting effect on mouse lymphocyte, and the cat B lymphocyte stimulating factor cDNA of the invention can be applied to producing recombinant cat B lymphocyte stimulating factor as cat immunopotentiator.

Description

Cat B lymphocyte stimulating factor cDNA and its coding protein, cloning method and use

Technical Field

The invention belongs to the field of biological genetic engineering, and particularly relates to a cat B lymphocyte stimulating factor cDNA, and a coding protein, a cloning method and application thereof.

Background

The B lymphocyte stimulating factor of the TNF family (BAFF, also known as BLyS, TALL-1, THANK, zTNF4 or TNFSF13B) is a transmembrane protein consisting of 285 amino acids, can function in membrane-bound and extracellular-soluble forms, and plays a major role in B cell survival, proliferation and differentiation. BAFF is a type II transmembrane protein that forms trimers with biological activity. BAFF interacts with three cell surface receptors of the TNF receptor superfamily (TNFRSF): BAFF receptors (BAFF-R, also known as BR3, CD268, and TNFRSF17), B cell maturation antigens (BCMA and TNFRSF13C), transmembrane activators and calcium modulators and cyclophilin ligands (TACI, CD267, and TNFRSF13B), all of which are predominantly expressed in B cells. BAFF is the only ligand for BAFB-R, while it shares receptor specificity for TACI and BCMA with proliferation-inducing ligand (APRIL). BAF-R, TACI and BCMA exhibit unique but overlapping expression patterns and functional analysis revealing the distinct roles of these three receptors in mediating BAFF (and APRIL) signaling, BAFB-R specifically binding to BAFF, which is the primary receptor triggering BAFF-induced B cell survival. Stimulation of BAFF-R can effectively activate NF-kB 2 pathway, which provides basis for BAFF action on B cell survival. The crystal structure of the functionally soluble part of human baff (hsbaff) shows that it has an exceptionally long D-E loop, forming a region that may be important for receptor binding and viroid assembly, compared to other TNF family members. BAFF plays an important role in B cell maturation, survival and T cell activation as well as autoimmune diseases. Insufficient levels of BAFF will fail to activate B cells to produce sufficient immunoglobulin and cause immunodeficiency. As an immunostimulant, BAFF is essential for maintaining normal immunity.

According to the results of three international major nucleic acid sequence databases of GenBank, EMBL and DDBJ, BAFF cDNA of human, mouse, pig, cow, sheep, rabbit, chicken, duck and goose has been cloned and applied for sequence numbers in GenBank database respectively, wherein the sequence numbers are AF116456, AF119383, EF041845, EU213012, FJ793192, EF202603, AF506010, DQ445092 and DQ 874394. The feline B lymphocyte stimulating factor (cBAFF) cDNA has not been cloned yet, and studies on feline BAFF genes are still in a complete gap state throughout China and abroad.

Cats are receiving increasing attention as domestic pets around the world. It is important to improve the immunity of cats, especially those with rare pets. In recent years, with the continuous deep research on cytokines and the development of molecular biology technology, a large number of cytokines can be cloned and recombined to express, and a plurality of cytokine genetic engineering drugs are marketed, so that a new means is provided for treating diseases, and great economic and social benefits are created, therefore, the recombined cBAFF protein has potential great market application value.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides cDNA of a feline B lymphocyte stimulating factor (cBAFF) extracted from a cat, the invention clones a cBAFF long cDNA sequence from the cat, researches the expression profile of the cBAFF long cDNA sequence, recombines and expresses to generate csBAFF protein with biological activity, and determines the biological activity of the csBAFF protein.

The invention also provides a protein coded by the cDNA of the cat B lymphocyte stimulating factor, a cloning method and application.

The technical scheme is as follows: in order to achieve the purpose, the base sequence of the cat B lymphocyte stimulating factor cDNA is shown as SEQ ID NO. 1.

The amino acid sequence of the protein coded by the cDNA of the cat B lymphocyte stimulating factor is SEQ ID NO. 2.

The cloning method of the cDNA of the cat B lymphocyte stimulating factor comprises the following steps:

(1) extracting total RNA of the spleen of the cat by a TRIzol method;

(2) designing a degenerate primer according to the conserved sequence of the B lymphocyte stimulating factor;

(3) amplifying a P1 segment by an RT-PCR method, and judging the amplified segment to be a BAFF gene family by carrying out homology analysis by using BLAST software;

(4) specific primers were designed based on the amplified P1 fragment, and the full-length BAFF cDNA was cloned by RACE PCR.

Wherein the base sequences of the degenerate primers cBAFF1 and cBAFF2 in the step (2) are shown in SEQ ID NO. 3-4;

sense oligonucleotide primer cBAFF1(SEQ ID No. 3):

5'-GTCNCCTGTNCAGNGTGGNCNTCCTGC-3'；

antisense oligonucleotide primer cBAFF2(SEQ ID No. 4):

5'-GGANCAANTTCNCCAGNCTCANTTCGT-3'；

wherein the specific primer specific primers 3'GSP1 and 3' GSP2, 5'GSP1 and 5' GSP2 in the step (4) have base sequences shown in SEQ ID NO. 5-8;

3'GSP1(SEQ ID NO.5)：5'-TAAAGCGGGTCCTGCTATTCAAGTTC-3’；

3'GSP2(SEQ ID NO.6)：5'-TCTGGGTACCGCAA AAGCATTAGG-3'；

5'GSP1(SEQ ID NO.7)5'-AGGGCCCGGCTCAGCCCGAAGCTCGCG-3'；

5'GSP2(SEQ ID NO.8)5'-GCTCCCCAGCTCGGCTTGCAGGACGG-3'。

further, primers CB-1 and CB-2 are designed after cloning the full-length BAFF cDNA in the step (4). The full-length cat cBAFF is expanded, and the sequences of the CB-1 and the CB-2 are shown in SEQ ID NO. 9-10;

CB-1(SEQ ID NO.9):5'-ATGGAGGGCTGCGCGGAAGGCGA GCG-3'；

CB-2(SEQ ID NO.10):5'-TCACAGAAGTTTCAATGCTCCAAAA-3'。

the invention relates to a recombinant vector pSUMO-csBAFF of an extracellular soluble region of cat B lymphocyte stimulating factor cDNA.

The invention relates to a fusion protein csBAFF expressed by a recombinant vector of an extracellular soluble region.

The application of the cat B lymphocyte stimulator extracellular soluble region protein csBAFF produced by the cat B lymphocyte stimulator cDNA and recombined as a cat immunopotentiator.

The invention firstly extracts total RNA from spleen tissues of cats, utilizes an RT-PCR method to clone a full-length BAFF cDNA sequence of the cats by a homologous cloning (homology cloning) strategy and a cDNA terminal rapid amplification method (RACE).

The specific operation is as follows:

(1) method for extracting total RNA of spleen tissue of cat by using Trizol reagent one-step method

(2) Analyzing the conserved regions of BAFF cDNA of human, mouse and cattle by Clustal w software, and designing a degenerate primer cBAFF 1; cBAFF2 was PCR amplified to give a 530bp fragment P1.

(3) Homology analysis was performed on the fragment P1 using BLAST software to determine whether the amplified fragment belongs to the BAFF gene family.

(4) Designing 3' RACE specific primers 3' GSP1 and 3' GSP2 by using the obtained homologous cloning fragments; 5' RACE specific primers 5' GSP1 and 5' GSP 2. 3'RACE amplification results in a3' terminal fragment P2 (including 3'UTR) of the BAFF full-length cDNA, and 5' RACE amplification results in a 5 'terminal fragment P3 (including 5' UTR) of the BAFF full-length cDNA. The three sequences of P1, P2 and P3 are spliced by BLAST2 software to obtain the BAFF full-length cDNA.

(5) Primers CB-1 and CB-2 are designed at the 5 'end and 3' end respectively, and the full-length cBAFF is expanded.

Similarity search is carried out on the obtained sequence cBAFF and GenBank database sequences, the similarity rate of the cat BAFF to the known human, mouse, cow and dog BAFF sequences is higher and is respectively 88.9%, 67.9%, 94.1% and 93.5%, and the sequence can be determined to be full-length cDNA of cat B lymphocyte stimulating factor (cBAFF), and the open reading frame of the cDNA is shown as SEQ ID NO. 1.

In the present invention, full-length cBAFF cDNA is cloned, its expression profile is studied, biologically active csBAFF is produced, and its biological activity is determined. The study reports for the first time the in vitro characterization of feline BAFF, which can be used as a potential immune factor to enhance the immune efficacy of cats. Because the B lymphocyte stimulating factor (BAFF) has stronger immunity enhancing capability, the gene engineering cat BAFF protein is possibly developed into a biological agent capable of enhancing the immunity of cats so as to enhance the immunity of cats.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the cDNA of cat B lymphocyte stimulating factor (cBAFF) cloned from cat for the first time can be synthesized by an artificial method, and the gene is further researched: the expression of the cBAFF in each tissue (heart, liver, spleen, lung, intestine and kidney) through fluorescent quantitative PCR analysis shows that the expression of the cBAFF in all the detected tissues is the highest, the expression of the cBAFF in the spleen is the lowest, and the expression of the cBAFF in the small intestine is the lowest; these results indicate that BAFF plays an important role in the immune system of cats. The recombinant fusion protein of the gene has the high activity function of cBAFF; the protein coded by the gene has obvious survival/proliferation promoting effect on mouse lymphocyte.

The cDNA of the cat B lymphocyte stimulating factor (cBAFF) is recombined and applied by the existing genetic engineering method to produce the recombined and soluble cBAFF protein (csBAFF) which is used as an immunopotentiator of cats to enhance the immunity of the cats, and the gene sequence of the csBAFF protein can be transferred into animals by the existing genetic engineering method to enhance the immunity of the animals, so that the cDNA is applied to feeding.

Drawings

FIG. 1 is a diagram showing a base sequence of feline BAFF full-length cDNA (GenBank NO. MH151314) and the corresponding amino acid sequence (□) and (transported) marked with a stop codon TGA;

FIG. 2 is an analysis of the expression level of feline BAFF mRNA in each tissue. GAPDH as a reference scheme;

FIG. 3 shows the inducible expression of the fusion protein sumo-csBAFF in BL21(DE3), Ni⁺The SDS-PAGE identification of column purification and the western-blotting identification result of mouse anti-His 6-tag are shown in the figure; (in the figure, the band 1 is the whole mycoprotein without IPTG induction; 2 is the whole mycoprotein after IPTG induction for 4.5 hours; 3 is the Ni⁺Purified target protein; 4 is protein csBAFF after enzyme digestion by sumo protease; 5 is with mouse anti-His₆Western-blotting identification result of monoclonal antibody

FIG. 4 is a graph showing the results of testing csBAFF for the pro-survival effect on mouse lymphocytes in vitro by MTT cytotoxicity assay; (PBS is blank control, 10. mu.g/ml csBAFF, 2. mu.g/ml anti-mouse IgM are negative controls)

FIG. 5 is a schematic representation of flow cytometry to detect the pro-survival effect of csBAFF on mouse lymphocytes; the left side is a blank control with PBS alone and the right side is the assay results after 48 hours of treatment with 10mg/ml csBAFF.

Detailed Description

The invention is further illustrated by the following figures and examples.

Example 1

8 months old domestic cats; farm purchased from Nanjing red sun

(1) Designing a primer: the conserved regions of human, murine and bovine BAFF cDNA are analyzed by Clustal w software, and a degenerate primer cBAFF1 is designed: 5 '-GTCNCCTGTNCAGNGTGGNCNTCCTGC-3'; cBAFF 2: 5'-GGANC AANTTCNCCAGNCTCANTTCGT-3'.

(2) Extracting total RNA: total RNA of feline spleen cells was extracted using an RNA extraction reagent TRIzol (Invitrogen corporation) according to the manual, and the quality and purity thereof were identified by formaldehyde-denatured agarose gel electrophoresis, and the concentration thereof was measured with an ultraviolet spectrophotometer.

(3) RT-PCR: PCR amplification was carried out using cBAFF1 and cBAFF2 as primers to obtain 530bp fragment P1.

Reverse transcription

Total RNA extracts were added sequentially to DEPC-treated 1.5ml Eppendorf tubesExtract 3. mu.l, Oligod (T)₁₈Mu.l of 10mmol/L dNTP 2. mu.l and 5. mu.l of DEPC water are placed; ice-bath is carried out immediately for 5min after 15min at 65 ℃; adding 4 mul of 5 XTRT reaction buffer solution, 0.5 mul (20u) of RNase inhibitor, 2 mul of AMV reverse transcriptase and 2.5 mul of DEPC water into the system, reacting for 1.5h at 42 ℃, stopping the reaction at 94 ℃ for 10min, and placing on ice;

②RT-PCR

PCR was performed using the template obtained by reverse transcription in a 50. mu.l system with 2.5. mu.l each of 10. mu. mol/L upstream and downstream primers, 4. mu.l of 2.5mmol/L dNTP, 25mmol/L MgCl ₂3 μ l, 5 μ l of 10 Xbuffer, 5 μ l cDNA template, 1 μ l rTaq enzyme, ddH₂O27. mu.l. The reaction procedure is as follows: 94 ℃ for 5min, 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 1min, 30 cycles, and finally 72 ℃ for 5 min.

After the reaction is finished, products are separated in 1% agarose gel by electrophoresis, DNA bands of about 530bp are obtained by recovery of a gel recovery kit, the DNA bands are cloned into a pMD18-T vector, transformants are screened by agarose plates containing Amp antibiotics (50mg/ml), and positive clones are picked out and sent to Shanghai Yingjun sequencing company for base sequence determination.

(4) The cDNA terminal rapid amplification method (RACE) clones the cDNA sequence of cat full-length BAFF. The resulting fragment P1 was used to design 3 '-RACE specific primers (3' -GSP 1: 5'-TAAAGCGGGTCCTGCTATTCAAGTTC-3'; 3 '-GSP 2: 5'-TCTGGGTACCGCAAAAGCATTAGG-3') and 5' -RACE specific primers (5 '-GSP 1: 5'-AGGGCCCGGCTCAGCCCGAAGCTCGCG-3'; 5' -GSP 2: 5'-GCTCCCCAGCTCGGCTTGCAGGACGG-3'). 3' RACE amplification results in a terminal fragment P2 (including 3' UTR) of BAFF full-length cDNA3, and 5' RACE amplification results in a terminal fragment P3 (including 5' UTR) of BAFF full-length cDNA 5 '. The three sequences of P1, P2 and P3 are spliced by BLAST2 software to obtain the BAFF full-length cDNA. Primers CB-1(5'-ATGGAGGGCTGCGCGGAAGGCGAGCG-3') and CB-2(5'-TCACAGAAGTTTCAATGCTCCAAAA-3') are designed at the 5 'end and the 3' end respectively to expand the full-length cBAFF, corresponding amino acid sequences are aligned, and the system relationship and the molecular structure are subjected to bioinformatics analysis.

(5) And (3) homologous retrieval: similarity search and homology analysis are carried out on the obtained cDNA sequence in three main nucleic acid sequence databases of GenBank, EMBL and DDBJ, the sequence is found to have high similarity with the known human, mouse, cow and dog amino acid sequences, namely 88.9 percent, 67.9 percent, 94.1 percent and 93.5 percent respectively, the sequence can be determined to be the full-length cDNA of cat B lymphocyte stimulating factor (cBAFF), the base sequence of the full-length cDNA is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

SEQ ID NO.1：

ACGCGGGGCGGGATGCAGCTGGGCAGACGCGGGGAGACCCCGGGGAGCTCTCCCAGGGGCCAAGGCACAGCCAGGCCATGTAGTCGGTGTGGGACCTCAGCAAGCACCGCTCACAGGAAATGATCCCCTCCCTGCGGCCACTTTCTCGAAAGGCCTGGACCTTCCAAGTCCCCCGAGTGCCATGGAGGGCTGCGCGGAAGGCGAGCGGTCACGCCCTCGTCCCCGCCCGGAGGGGGGAGAAGAAATGAAAGTGAAGGAGCCACCCGGCTCGCGCCCCAAAGACGCAGCGCTGCTGGCCGTGACCCTGCTGCTGGCGCTCACGTCCTGCTGCCTGTCGGTGGTGTCCCTGTGCAGGGTGGCCGTCCTGCAAGCCGAGCTGGGGAGCCTGCGGGCGGAGCTGCGGGGGCCCCGGGAGCCCCGCGTGCTTCGGGCTGAGCCGGGCCTCCCCCGTCGGGGGCCGCAACCCCCGGGGCGTCCGGCGCGCGCCTGGCCCCCGCGGTCCCCAGCGCGCTGAAAGGGATCTTCGCACCAACCCCGGCGGGAGAGAGCAACTCCAGCCAGAGCGGCAGAAGGAAGCGCGCCGCTCTGGATCCCGAAGACACAGTCATTCAAGACTGCTTGCAACTGATCGCAGACAGCGACACACCTACTATACGAAAAGGAGCCTACACGTTCGTTCCGTGGCTGCTCAGCTTTAAAAGAGGAAGGGCCCTGGAAGAAAAGGAAAATAAAATCCTGGTGAAAGAAACCGGTTACTTCTTTATATACGGGCAGGTTTTATACACGGATAACACCTTTGCTATGGGACACCTAATACAGAGGAAAAAAGTCCACGTCTTTGGGGACGAACTGAGTCTGGTGAATTTGTTCCGATGTATTCAAAATATGCCTGAAACACTACCCAATAATTCCTGTTATTCCGCTGGCATCGCAAAGCTGGAAGAAGGAGATGAACTCCAACTGGCAATACCGCGGGAAGACGCTAAAATATCGCAAGATGGAGATGGCACGTTTTTTGGAGCATTGAAACTTCTGTGACCTACTTCTACCTTGTTCGCGGCGAATTCCTTCCTTTCTCTGTGCCTCTAAGGAGAGAACACTTCACTGGAAATGCCAGAGGAAGAAAACCAGGAGTTACCGAACTCTTTTCTGTGAGCTATTTGTTTTGATTTGCTGAACCTAGAGCAAAACAGGAAATTGAACAGACAGTCGCAGCCCAGGGGTGTCATGTGAATTACAAGAAATAGAACCCGTTTGAGGAAAGATAGAAGTAGCCCTCTCCCTATAAAAGCCACGTTGAGCAACTTAGGCATCGCGGCTTGGCTGGTAGCAAACACAGGTTTCCAAGGGGCGATAATCCTATTTCTTCCTAAACAGCATTCCGTTGTGCGCCGGGGAACAAGTGCCCATGTCCGCAGGACCTTAACTGAGACATTTTGGGAAATCTCAGGATTCATCCCCTCTATGTTAAATATGCCCCCCCCCACCTCTTCCAGTTAACGTTGTAGGAAGTAAGAAAAAAAAAAAA

SEQ ID NO.2：

MEGCAEGERSRLRPRPERGEEMKVKEPPGSRPKDAALLAVTLLLALTSCCLSVVSLCRVAVLQAELGSLRAELRGPREPRELRAEPGPPPSGAATPGASGARLAPAVPSALKGIFAPTPAGESNSSQSGRRKRAALDPEDTVIQDCLQLIADSDTPTIRKGAYTFVPWLLSFKRGRALEEKENKILVKETGYFFIYGQVLYTDNTFAMGHLIQRKKVHVFGDELSLVNLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPREDAKISQDGDGTFFGALKLL*

Example 2

Analysis of expression level of feline BAFF gene (feline B lymphocyte stimulating factor cDNA sequence) in each tissue:

using the method for extracting RNA in example 1, respectivelyCat (cat)Total RNA of heart (heart), liver (liver), spleen (spleen), lung (lung) and intestine (intestine) is subjected to reverse transcription by reverse transcriptase to form cDNA, GAPDH is used as an internal reference, and the expression level of BAFF gene of cats in each tissue is researched by using a real time-PCR method.

The amplification primer of the target gene is

Q1(SEQ ID NO.11)：5'-CGGGCAGGTTTTATACACGG-3'；

Q2(SEQ ID NO.12)：5'-GATGCCAGCGGAATAACAGG-3'；

The amplification primer of GAPDH is

Q5(SEQ ID NO.13)：5'-CATTGCCC TCAACGACCACTTTGTC-3'；

Q6(SEQ ID NO.14)：5’-CTCCTTGGAGGCCATGTGGACCATG-3'；

Three replicates of each sample were run with DEPC water as a blank. The reaction system is as follows: mix 12.5. mu.l, H₂O9.5. mu.l, Primer 11. mu.l, Primer 21. mu.l, and templete (amplified template sequence cat cDNA) 1. mu.l, and amplification primers for the target gene and amplification primers for GAPDH were simultaneously amplified, respectively. The reaction procedure is as follows: 95 ℃/3min, 40cycles (95 ℃/30s, 60 ℃/1 min). Setting a fluorescence detection threshold (threshold) at an appropriate position in the exponential growth region of the amplification curve for each sample will result in a Ct value at the intersection of the threshold and the amplification curve, where the Ct value is the number of cycles (threshold cycle) at which the threshold is reached, and is linearly inversely proportional to the logarithm of the number of initial templates. The difference between the Ct value of each target gene and the Ct value of the corresponding GAPDH is delta Ct, the difference between the delta Ct value of each sample and the delta Ct value of the sample with the lowest expression level is delta Ct, and the relative template number of cBAFF in each sample can be expressed as 2^-ΔΔCt。

As shown in FIG. 2, cBAFF is mainly expressed in immune organs such as spleen, and is expressed in the small intestine in the lowest amount.

Constructing an extracellular soluble region (csBAFF) recombinant expression vector of cat BAFF and carrying out induced expression on the recombinant expression vector in escherichia coli:

based on the cDNA sequence of cat BAFF and the stuI cleavage site of pSUMO, a pair of specific PCR primers P1(SEQ ID NO.13) (5'-CGCCGCTCTGGATCCCGAAGACACAGTCA-3') and P2(SEQ ID NO.14) (5'-CCCAAG CTTCAGAAGTTTCAATGCTCCAAAAAA-3') were designed, and a BamHI cleavage site was inserted into the downstream primer. The PCR system using cat BAFF gene (cat B lymphocyte stimulating factor cDNA sequence) as template is 50 μ L, 10 μmol/L upstream and downstream primers are 2.5 μ L each, 2.5mmol/L dNTP 4 μ L, 25mmol/L MgCl ₂3 μ l, 5 μ l of 10 Xbuffer, 5 μ l cDNA template, 1 μ l rTaq enzyme, ddH₂O27. mu.l. The reaction procedure is as follows: carrying out 30 cycles of 94 ℃ for 5min, 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 1min, and finally carrying out incubation at 72 ℃ for 5min, tapping and recovering a PCR product, carrying out enzyme digestion on the recovered product by Stu1 and BamH I, carrying out enzyme digestion on a SUMO vector only by BamH I, connecting the enzyme digested vector and the PCR product by T4 DNA Ligase, and transforming the connected product into E.coli top10 to obtain a recombinant strain containing pSUMO-csBAFF; selecting a positive recombinant strain, carrying out expanded culture to extract a pSUMO-csBAFF recombinant plasmid, and constructing to obtain a recombinant vector pSUMO-csBAFF. The recombinant vector is transferred into an escherichia coli expression strain BL21(DE3), and the target protein SUMO-csBAFF can be expressed in cytoplasm in a high-solubility mode. SDS-PAGE detection (FIG. 3) showed that the size of the fusion protein was approximately 37.5 kDa. After excision of the tag SUMO with SUMO protease, a csBAFF protein of approximately 17.5kDa is obtained.

Ni + affinity purification of recombinant protein of interest:

IPTG (final concentration of 0.2mmol/L in bacterial liquid) low-temperature induction of Escherichia coli expression strain BL21(DE3) containing recombinant vector pSUMO-csBAFF for 48 hours, collecting bacteria at 4 ℃, ultrasonically crushing (160W, working for 4s, pausing for 8s, totally 99 times) expression bacteria on ice, centrifuging and ultrasonically crushing liquid at 4 ℃, 13, 000 Xg and 20min, discarding precipitate, and reserving supernatant Ni⁺-an NTA column. The process is as follows: 3 volume Binding Buffer balance Ni⁺After NTA columnThe supernatant containing the soluble recombinant protein was manually loaded, washed with 10 volumes of Binding buffer followed by 6 volumes of Washing buffer (20mmol/L Imidazole, 0.5mol/L NaCl, 20mmol/L LTris HCl, pH 7.9) to remove the contaminating protein, and finally eluted with Elute buffer (1mol/L Imidazole, 0.5mol/L LNaCl, 20mmol/L TrisHCl, pH 7.9) to Elute the target protein, collected in tubes, and desalted with PBS (pH 7.4). SDS-PAGE detects the protein purity of each collection tube and an ultraviolet spectrophotometer determines the protein concentration. As shown in FIG. 3 (lane 3), the target protein obtained by expression was subjected to affinity purification using Ni + column to obtain active target protein with a purity of 95%, and the target protein was collected, dialyzed, filtered and sterilized, and then stored.

Western blot analysis:

the primary antibody used in Western-blot is mouse anti-His₆The monoclonal antibody and the secondary antibody are goat anti-mouse IgG marked by horseradish peroxidase. The method comprises the following brief steps: the method comprises the steps of firstly carrying out SGS-PAGE on a sample, then electrically transferring protein onto a nitrocellulose membrane (NC membrane) by an immersion method, marking each strip of a protein marker by using a marker pen, then sequentially sealing the strip for 1h at room temperature by 5% skimmed milk powder, incubating the strip with primary antibody for 1h, incubating the strip with HRP-marked secondary antibody IgG for 1h, strictly washing the membrane after each step is finished, and finally adding TMB to carry out light-shielding color development. As a result, a specific band appeared at the target protein position as shown in FIG. 3 (band 5).

csBAFF effect on mouse lymphocyte survival promotion experiment:

(1)MTT

shearing mouse spleen into small plate containing PBS solution, grinding, sieving with 200 mesh sieve, washing with PBS for several times, removing red blood cells, adding lymphocyte separation liquid, and separating to obtain mouse lymphocyte. The isolated mouse lymphocytes were adjusted to a cell concentration of about 5X 10 using RPMI 1640 (containing 10% FCS, 100U/ml penicilin/streptomycin) medium⁶At one/ml, 50. mu.l of cells, 150. mu.l of RPMI 1640 medium, were added to each well of a 96-well plate. Sequentially adding recombinant protein csBAFF in groups, wherein the final concentrations of the recombinant protein are respectively 2, 4, 6, 8, 10, 12 and 14 mu g/ml, and the final concentrations of the anti-IgM are respectively 2 mu g/ml; PBS was used as a blank control, and 10. mu.g/ml csBAFF and 2. mu.g/ml anti-mouse IgM were used as negative controls. At 37 deg.C, 5% CO₂Culturing under the condition of concentrationAfter 48h of incubation 50. mu.l of 2mg/mL MTT was added to each well at 37 ℃ with 5% CO₂Continuously culturing for 4h, centrifuging, removing supernatant, adding 150 μ l DMSO microporous plate, shaking for 10min to dissolve crystals, and determining OD of each well₄₉₀The value is obtained. Three sets of duplicate wells were set up for the experiment, the mean was taken and the standard error was calculated. As shown in FIG. 4, the csBAFF cloned in the experiment has obvious survival promoting effect on mouse lymphocytes and shows dose-dependent effect.

(2) Flow cytometry FCM

Shearing mouse spleen into small plate containing PBS solution, grinding, sieving with 200 mesh sieve, washing with PBS for several times, removing red blood cells, adding lymphocyte separation liquid, and separating to obtain mouse lymphocyte. The isolated mouse lymphocytes (5X 10)⁵) With 10mg/ml csBAFF at 37 ℃ with 5% CO₂After 48h incubation, three washes were performed with PBS, followed by staining with 2.5mg/ml PI at 37 ℃ for 30min and three washes with PBS. Detection was performed by flow cytometry (BD bioseicces). PBS was added as a blank control. As shown in FIG. 5, the fusion protein csBAFF can promote the survival of mouse lymphocytes under the co-stimulation effect of anti-mouse IgM compared with the control.

Example 3

The cat B lymphocyte stimulator cDNA obtained in example 1 was used to produce recombinant cat BAFF as a cat immunopotentiator by a conventional genetic engineering method.

Example 4

The cDNA of the cat B lymphocyte stimulating factor obtained in the example 1 is transferred into an animal body by the existing gene engineering method to increase the immunity of the animal body, and is applied to feeding.

The method according to the present invention can modify the feline B-lymphocyte stimulator gene by the existing genetic engineering techniques and be used for research and production.

Sequence listing

<110> university of Nanjing university

<120> cat B lymphocyte stimulating factor cDNA and coding protein, cloning method and application thereof

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<170> SIPOSequenceListing 1.0

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<213> Cat B lymphocyte stimulating factor cDNA (cBAFF)

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acgcggggcg ggatgcagct gggcagacgc ggggagaccc cggggagctc tcccaggggc 60

caaggcacag ccaggccatg tagtcggtgt gggacctcag caagcaccgc tcacaggaaa 120

tgatcccctc cctgcggcca ctttctcgaa aggcctggac cttccaagtc ccccgagtgc 180

catggagggc tgcgcggaag gcgagcggtc acgccctcgt ccccgcccgg aggggggaga 240

agaaatgaaa gtgaaggagc cacccggctc gcgccccaaa gacgcagcgc tgctggccgt 300

gaccctgctg ctggcgctca cgtcctgctg cctgtcggtg gtgtccctgt gcagggtggc 360

cgtcctgcaa gccgagctgg ggagcctgcg ggcggagctg cgggggcccc gggagccccg 420

cgtgcttcgg gctgagccgg gcctcccccg tcgggggccg caacccccgg ggcgtccggc 480

gcgcgcctgg cccccgcggt ccccagcgcg ctgaaaggga tcttcgcacc aaccccggcg 540

ggagagagca actccagcca gagcggcaga aggaagcgcg ccgctctgga tcccgaagac 600

acagtcattc aagactgctt gcaactgatc gcagacagcg acacacctac tatacgaaaa 660

ggagcctaca cgttcgttcc gtggctgctc agctttaaaa gaggaagggc cctggaagaa 720

aaggaaaata aaatcctggt gaaagaaacc ggttacttct ttatatacgg gcaggtttta 780

tacacggata acacctttgc tatgggacac ctaatacaga ggaaaaaagt ccacgtcttt 840

ggggacgaac tgagtctggt gaatttgttc cgatgtattc aaaatatgcc tgaaacacta 900

cccaataatt cctgttattc cgctggcatc gcaaagctgg aagaaggaga tgaactccaa 960

ctggcaatac cgcgggaaga cgctaaaata tcgcaagatg gagatggcac gttttttgga 1020

gcattgaaac ttctgtgacc tacttctacc ttgttcgcgg cgaattcctt cctttctctg 1080

tgcctctaag gagagaacac ttcactggaa atgccagagg aagaaaacca ggagttaccg 1140

aactcttttc tgtgagctat ttgttttgat ttgctgaacc tagagcaaaa caggaaattg 1200

aacagacagt cgcagcccag gggtgtcatg tgaattacaa gaaatagaac ccgtttgagg 1260

aaagatagaa gtagccctct ccctataaaa gccacgttga gcaacttagg catcgcggct 1320

tggctggtag caaacacagg tttccaaggg gcgataatcc tatttcttcc taaacagcat 1380

tccgttgtgc gccggggaac aagtgcccat gtccgcagga ccttaactga gacattttgg 1440

gaaatctcag gattcatccc ctctatgtta aatatgcccc cccccacctc ttccagttaa 1500

cgttgtagga agtaagaaaa aaaaaaaa 1528

<210> 2

<211> 285

<212> PRT

<213> Cat B lymphocyte stimulating factor (cBAFF)

<400> 2

Met Glu Gly Cys Ala Glu Gly Glu Arg Ser Arg Leu Arg Pro Arg Pro

1 5 10 15

Glu Arg Gly Glu Glu Met Lys Val Lys Glu Pro Pro Gly Ser Arg Pro

20 25 30

Lys Asp Ala Ala Leu Leu Ala Val Thr Leu Leu Leu Ala Leu Thr Ser

35 40 45

Cys Cys Leu Ser Val Val Ser Leu Cys Arg Val Ala Val Leu Gln Ala

50 55 60

Glu Leu Gly Ser Leu Arg Ala Glu Leu Arg Gly Pro Arg Glu Pro Arg

65 70 75 80

Glu Leu Arg Ala Glu Pro Gly Pro Pro Pro Ser Gly Ala Ala Thr Pro

85 90 95

Gly Ala Ser Gly Ala Arg Leu Ala Pro Ala Val Pro Ser Ala Leu Lys

100 105 110

Gly Ile Phe Ala Pro Thr Pro Ala Gly Glu Ser Asn Ser Ser Gln Ser

115 120 125

Gly Arg Arg Lys Arg Ala Ala Leu Asp Pro Glu Asp Thr Val Ile Gln

130 135 140

Asp Cys Leu Gln Leu Ile Ala Asp Ser Asp Thr Pro Thr Ile Arg Lys

145 150 155 160

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

165 170 175

Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr

180 185 190

Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Asn Thr Phe Ala Met

195 200 205

Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu

210 215 220

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

225 230 235 240

Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly

245 250 255

Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asp Ala Lys Ile Ser Gln

260 265 270

Asp Gly Asp Gly Thr Phe Phe Gly Ala Leu Lys Leu Leu

275 280 285

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gtcncctgtn cagngtggnc ntcctgc 27

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggancaantt cnccagnctc anttcgt 27

<210> 5

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

taaagcgggt cctgctattc aagttc 26

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tctgggtacc gcaaaagcat tagg 24

<210> 7

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agggcccggc tcagcccgaa gctcgcg 27

<210> 8

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gctccccagc tcggcttgca ggacgg 26

<210> 9

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggagggct gcgcggaagg cgagcg 26

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcacagaagt ttcaatgctc caaaa 25

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cgggcaggtt ttatacacgg 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gatgccagcg gaataacagg 20

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cattgccctc aacgaccact ttgtc 25

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ctccttggag gccatgtgga ccatg 25

Claims

1. A cDNA of cat B lymphocyte stimulating factor is characterized in that the base sequence is shown in SEQ ID NO. 1.

2. The protein encoded by the feline B lymphocyte stimulator cDNA of claim 1, having the amino acid sequence of SEQ ID No. 2.

3. A method for cloning a cDNA of a feline B-lymphocyte stimulating factor according to claim 1, which comprises the following steps:

(1) extracting total RNA of the spleen of the cat by a TRIzol method;

(4) specific primers were designed based on the amplified P1 fragment, and cDNA of cat full-length BAFF was cloned by RACE PCR.

4. The cloning method of claim 3, wherein the degenerate primers of step (2) are cBAFF1 and cBAFF2, and the base sequences are shown in SEQ ID NO. 3-4.

5. The cloning method of claim 3, wherein the specific primers in step (4) are 3'GSP1, 3' GSP2, 5'GSP1 and 5' GSP2, and the base sequences thereof are shown in SEQ ID NO. 5-8.

6. The cloning method according to claim 3, wherein primers CB-1 and CB-2 are designed after cloning the full-length BAFF cDNA in the step (4) to expand the full-length feline cBAFF, and the sequences of the CB-1 and CB-2 are shown in SEQ ID NO. 9-10.

7. A recombinant vector pSUMO-csBAFF comprising a base sequence encoding an extracellular soluble region of the feline B lymphocyte stimulator cDNA according to claim 1, wherein the specific PCR amplification primers of said recombinant vector pSUMO-csBAFF are P1 and P2, and the base sequences thereof are SEQ ID No.13 and SEQ ID No.14, respectively.

8. A fusion protein SUMO-csBAFF expressed using the recombinant vector comprising a base sequence encoding an extracellular soluble region of a feline B lymphocyte stimulator cDNA according to claim 7.

9. The use of the cat B lymphocyte stimulator cDNA and the recombinant cat B lymphocyte stimulator extracellular soluble domain protein csBAFF produced from the same as claimed in claim 1 in preparing a cat immunopotentiator.