CN113461826A

CN113461826A - Application of signal peptide in recombinant porcine interferon and thymosin fusion protein expression system

Info

Publication number: CN113461826A
Application number: CN202010241813.5A
Authority: CN
Inventors: 罗弟祥; 高小平; 代燕平
Original assignee: Chengdu Tongqien Biotechnology Co ltd
Current assignee: Chengdu Tongqien Biotechnology Co ltd
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2021-10-01

Abstract

The invention discloses an application of a signal peptide in enhancing the high-efficiency expression of recombinant porcine interferon and thymosin fusion protein. The signal peptide is selected from a feline interferon omega signal peptide comprising 23 amino acid sequences that direct the efficient expression of a fusion protein of porcine interferon alpha 1 and thymosin alpha 1(poIFN alpha 1-T alpha 1) in Chinese Hamster Ovary (CHO) cells. Compared with the signal peptide of the porcine interferon alpha 1(poIFN alpha 1), the secretory expression of the fusion protein of the recombinant porcine interferon alpha 1 carrying the feline interferon omega signal peptide and the thymosin alpha 1(rpoIFN alpha 1-T alpha 1) in CHO cells is obviously improved.

Description

Application of signal peptide in recombinant porcine interferon and thymosin fusion protein expression system

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of a feline interferon omega signal peptide in a fusion protein expression system of recombinant porcine interferon alpha 1 and thymosin alpha 1.

Background

Porcine interferons (poIFNs) are secreted proteins comprising about 18 to 30 amino acids at their N-terminusLetter Peptide numberThe newly synthesized IFNs enter the lumen of the endoplasmic reticulum under the guidance of the signal peptide, the signal peptide is cut off by the action of the signal peptidase, and the interferon is secreted out of the cell. The signal peptide carried by the protein is used for guiding the secretory expression of the interferon, and the protein has the advantages of having a reliable cutting site and being capable of obtaining a natural N terminal. However, in the expression system of the recombinant fusion protein, especially in different expression host cells, the signal peptide carried by itself may not exert the best effect of guiding secretion. Such as in Kun YinThe cell expresses porcine interferon alpha, and secretory protein can not be effectively obtained by using the signal peptide of the cell, but the original signal peptide of the porcine interferon alpha is replaced by introducing the melittin signal peptide which can be identified by the insect cell, so that the secretory expression of the porcine interferon alpha in the insect cell can be realized. The applicant of the present invention finds in research that high-efficiency expression and secretion of poIFN α 1 in mammalian cells cannot be obtained by using self signal peptide, but high-efficiency expression and secretion of poIFN α 1 in mammalian cells can be achieved by replacing other interferon signal peptides derived from mammals, such as replacing cat interferon ω signal peptide, which is presumed to be related to signal peptide carried by cat interferon ω, but there is no application example of guiding pig interferon expression in mammalian cells by cat interferon ω signal peptide so far.

Thymosin alpha 1(T alpha 1) is a bioactive polypeptide consisting of 28 amino acids, and has no species specificity. T alpha 1 has immunoregulation and antiviral effects, although with interferon similar efficacy, but the mechanism of action is totally different, therefore, clinical application of T alpha 1 and human interferon in combination has been proved to be better than each alone curative effect, show that the two combination has synergistic and complementary effects.

The invention replaces the signal peptide carried by the porcine interferon alpha 1 with the feline interferon omega signal peptide, the thymosin alpha 1 is placed at the C end of the porcine interferon alpha 1 through the connecting peptide with the length of 10 amino acids, the fusion protein of the recombinant porcine interferon alpha 1 and the thymosin alpha 1(rpoIFN alpha 1-T alpha 1) is expressed in CHO cells, and compared with the signal peptide, the secretory expression of the fusion protein of the poIFN alpha 1-T alpha 1 is obviously improved under the guidance of the feline interferon omega signal peptide.

Disclosure of Invention

The technical problem solved by the invention is to replace a poIFN alpha 1 signal peptide by a signal peptide of feline interferon omega, and realize the high-efficiency expression and secretion of the poIFN alpha 1 or the fusion protein of the poIFN alpha 1-T alpha 1 in CHO cells.

The invention is selected from a sharing signal peptide sequence of each subtype of feline interferon omega, replaces the self sequence of the poIFN alpha 1, and forms an amino acid sequence shown as

SEQ ID NO

1 and 2 and a nucleotide sequence shown as SEQ ID NO 2 and 4.

The invention also relates to the high-efficiency expression and secretion of the poIFN alpha 1-T alpha 1 fusion protein replaced by the signal peptide in CHO cells, which comprises the following steps:

(1) the signal peptide sequence of the poIFN alpha 1-T alpha 1 fusion protein gene is replaced by a cat interferon-omega signal peptide sequence, and poIFN alpha 1 containing self signal peptide is used as a reference, and is artificially synthesized and then recombined to an expression plasmid BP 001.

(2) Linearizing each expression plasmid and separately electrotransfering to CHO cells, preferably CHO DG44 cells, and after 48h serum-free culture, adding Methotrexate (MTX) to a final concentration of 10nM, followed by stepwise increase to 100 nM;

(3) cells with increasing MTX to 100nM were inoculated at the same density into shake flasks, continuously cultured in commercial medium and appropriately supplemented with nutritional supplements such as glucose. The supernatant was collected on the 9 th day of continuous culture, and the interferon-secretion amount in the supernatant was quantitatively determined by ELISA.

The invention has the beneficial effects that: the feline interferon omega signal peptide is used for replacing a porcine interferon signal peptide, and the poIFN alpha 1-T alpha 1 fusion protein is guided to realize high-efficiency secretory expression in a CHO DG44 cell, and the yield of the feline interferon omega signal peptide is obviously superior to that of the poIFN alpha 1-T alpha 1 carrying the signal peptide.

Drawings

FIG. 1 shows the Western blot results 48 hours after transfection of two expression plasmids

FIG. 2 compares the secretory expression levels of two poIFN α 1-T α 1 fusion proteins in CHO DG44 cells

The invention will be further illustrated by the following examples, without limiting its scope.

Example 1 Gene Synthesis and expression plasmid construction

Obtaining a porcine IFN-alpha 1 gene sequence from GenBank, wherein the accession number is as follows: NM-214393.1, replacing its signal peptide with the signal peptide sequence as SEQ ID NO 4, introducing endonuclease sites Avr II and Pac I at 5 'and 3' ends, respectively, and introducing GCCACC sequence before ATG, adding thymosin alpha 1 sequence at C-end of porcine IFN alpha 1, and adding 6 × His sequence at the end. The original gene sequence (p/poIFN alpha 1-T alpha 1) of the porcine interferon alpha 1 and thymosin alpha 1 fusion protein and the gene sequence (pFe/poIFN alpha 1-T alpha 1) of the porcine interferon alpha 1 and thymosin alpha 1 fusion protein which is replaced by the feline interferon-omega signal peptide are artificially synthesized and respectively recombined to pUC57 amplification plasmids. And further preparing two target gene fragments, respectively connecting the two target gene fragments to a BP001 expression vector, transforming Top10 competent cells, culturing the competent cells in an LB culture medium containing antibiotics, screening monoclonal colonies, extracting plasmid DNA, and performing double enzyme digestion identification to obtain expression plasmids p/poIFN alpha 1-T alpha 1 and pFe/poIFN alpha 1-T alpha 1.

Example 2 cell transfection and expression product identification

p/poIFN alpha 1-T alpha 1 and pFe/poIFN alpha 1-T alpha 1 expression plasmids are extracted by adopting an Axygen Plasmid Max Kit, and are linearized by PvuI endonuclease and then refer to Amaxa Cell Line Nucleofector^TMThe Kit V Kit shows that the linearized plasmid is stably transfected into CHO DG44 cells, supernatant is collected after 48 hours to identify the expression correctness of the product by western blot, the expression level difference of the transfected cells after 48 hours is compared (figure 1), and the obtained stably transfected cells are respectively named as S/poIFN alpha 1-T alpha 1 and SF/poIFN alpha 1-T alpha 1, and relevant information is shown in Table 1.

TABLE 1 Stable cell names of recombinant poIFN-alpha 1-T alpha 1 fusion proteins and related Signal peptide information

Example 3 comparison of expression yields of S/poIFN α 1-T α 1 and SF/poIFN α 1-T α 1 fusion proteins

The cells cultured 48h without serum were added with MTX containing 10nM and the culture was continued, followed by stepwise increase of MTX concentration to 100 nM. Cells with increasing MTX to 100nM were inoculated at the same density into shake flasks and cultured continuously for 9 days in commercial medium Acti Pro (cell density 5X 10 each)⁵cells/ml, 50ml volume) was added on days 3 and 6 of culture with 5% Cell Boost 7a/7b, and 1% glucose and 1% L-Glutamine were added in appropriate amounts. Terminating the culture on the 9 th day of culture andsupernatants from each culture were collected, and the expression levels of both fusion proteins were determined by quantifying the poIFN α 1-T α 1 fusion protein in the supernatants by ELISA. The results showed that the expression yield of SF/poIFN α 1-T α 1 fusion protein increased with time at 465mg/L, while the expression yield of S/poIFN α 1-T α 1 fusion protein also increased with time at about 184mg/L, but significantly lower than SF/poIFN α 1-T α 1 (FIG. 2).

The results show that compared with the signal peptide of the porcine interferon, the feline interferon omega remarkably promotes the secretion expression amount of the guiding poIFN alpha 1-T alpha 1 fusion protein in CHO cells. Therefore, the feline interferon omega signal peptide is more advantageous in realizing the industrialization of the expression of the fusion protein of the porcine interferon alpha 1 and the thymosin alpha 1 in mammalian cells, particularly in CHO cells.

Sequence listing

<110> Chengdong Qi En Biotech Ltd

<120> application of signal peptide in recombinant porcine interferon and thymosin fusion protein expression system

<130> 0

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> PRT

<213> signal peptide

<400> 1

Met Ala Leu Leu Leu Pro Leu Leu Thr Ala Leu Ala Leu Leu Thr Cys

1 5 10 15

Arg Pro Gly Gly Ser Leu Gly

20

<210> 2

<211> 70

<212> DNA

<213> signal peptide

<400> 2

atggccctgc tgctgcccct gctgaccgcc ctggccctgc tgacctgcag gcctggcggc 60

tccctgggct 70

<210> 3

<211> 235

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ala Leu Leu Leu Pro Leu Leu Thr Ala Leu Ala Leu Leu Thr Cys

1 5 10 15

Arg Pro Gly Gly Ser Leu Gly Cys Asp Leu Pro Gln Thr His Ser Leu

20 25 30

Ala His Thr Arg Ala Leu Arg Leu Leu Ala Gln Met Arg Arg Ile Ser

35 40 45

Pro Phe Ser Cys Leu Asp His Arg Arg Asp Phe Gly Ser Pro His Glu

50 55 60

Ala Phe Gly Gly Asn Gln Val Gln Lys Ala Gln Ala Met Ala Leu Val

65 70 75 80

His Glu Met Leu Gln Gln Thr Phe Gln Leu Phe Ser Thr Glu Gly Ser

85 90 95

Ala Ala Ala Trp Asn Glu Ser Leu Leu His Gln Phe Cys Thr Gly Leu

100 105 110

Asp Gln Gln Leu Arg Asp Leu Glu Ala Cys Val Met Gln Glu Ala Gly

115 120 125

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

130 135 140

Lys Tyr Phe His Arg Leu Thr Leu Tyr Leu Gln Glu Lys Ser Tyr Ser

145 150 155 160

Pro Cys Ala Trp Glu Ile Val Arg Ala Glu Val Met Arg Ser Phe Ser

165 170 175

Ser Ser Arg Asn Leu Gln Asp Arg Leu Arg Lys Lys Glu Gly Ser Gly

180 185 190

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Ala Ala Val Asp Thr

195 200 205

Ser Ser Glu Ile Thr Thr Lys Asp Leu Lys Glu Lys Lys Glu Val Val

210 215 220

Glu Glu Ala Glu Asn His His His His His His

225 230 235

<210> 4

<211> 705

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggccctgc tgctgcccct gctgaccgcc ctggccctgc tgacctgcag gcctggcggc 60

tccctgggct gcgacctgcc ccagacccac agcctggccc acaccagggc cctgcgcctg 120

ctggcccaga tgaggagaat cagccccttc agctgcctgg accaccgcag ggacttcggc 180

ttccctcagg aggccctggg cggcaaccag gtgcagaagg cccaggccat ggccctggtg 240

cacgagatgc tgcagcagac cttccagctg ttcagcaccg aggggagcgc cgccgcctgg 300

gacgagtccc tgctgcacca gttctgcacc ggcctggacc agcagctgag ggacctggaa 360

gcttgtgtga tgcaggaggc cggcctggaa ggcacacctc tactggagga agatagcatt 420

ctggccgtga gaaaatactt tcacagactg acactgtatc tgcaggagaa atcttatagc 480

ccttgtgctt gggaaattgt gagagctgaa gtgatgagag ccttttcttc tagtagaaat 540

ctgcaggata gactgagaaa aaaggaagga tccggcggag gaggatctgg aggaggagga 600

agctctgacg cagctgtgga cacaagctct gaaatcacca ctaaggatct gaaggaaaag 660

aaggaagtgg ttgaagaggc tgagaatcat caccaccatc atcac 705

Claims

1. An application of a signal peptide in a fusion protein expression system of recombinant porcine interferon and thymosin is characterized in that the signal peptide is selected from a feline interferon omega signal peptide.

2. The use of a signal peptide in an expression system of a fusion protein of recombinant porcine interferon and thymosin according to claim 1, wherein said fusion protein of recombinant porcine interferon and thymosin is a fusion protein of recombinant porcine interferon α 1 and thymosin α 1(rpoIFN α 1-T α 1).

3. The use according to claim 2, wherein the rpoIFN α 1-T α 1 fusion protein comprises the following operations:

(a) the signal peptide of the porcine interferon-alpha 1 is replaced by a feline interferon omega signal peptide, and the amino acid sequence of the signal peptide is shown as SEQ ID NO 1;

(b) thymosin alpha 1 was placed at the C-terminus of the fusion protein by a linker peptide of 10 amino acids in length, as shown in SEQ ID NO 3.

4. Nucleotide sequences encoding the signal peptide according to claim 3 and the fusion protein of porcine interferon alpha 1 and thymosin alpha 1, characterized in that said nucleotide sequences are represented by SEQ ID NO 2 and 4.

5. The use of a signal peptide according to claim 1 in a recombinant porcine interferon expression system, wherein said expression system is a mammalian cell expression system, including CHO cells, HEK 293 cells, NS0 cells, and the like.