WO2005117960A1

WO2005117960A1 - Sars dna vaccine and its preparing method, the use of spike gene of coronavirus for vaccine

Info

Publication number: WO2005117960A1
Application number: PCT/CN2004/000501
Authority: WO
Inventors: Yixin Zeng; Wenlin Huang; Jian Wang; Haide Tan; Peng Liu; Zhigang Pan; Qisheng Feng; Jiang Li; Lixi Huang; Miaohua Zhang; Lizhen Chen
Original assignee: Cancer Center Sun Yat-Sen University
Priority date: 2004-06-04
Filing date: 2004-06-04
Publication date: 2005-12-15

Abstract

The present invention belongs to the field of biological genetic engineering, relates to severe acute respiratory syndrome (SARS) DNA vaccine, its preparing method, and the use of spike gene of SARS coronavirus for SARS vaccine. The present invention is a kind of DNA vaccine, that is to extract the spike gene, clone into pcDNA3, then amplify, purify and prepare for DNA vaccine. To traditional live activated vaccine of virus, the present invention has more safety and immunogenicity, can effectively induce antibody in organism and protect organism against virus infection. It has substantial potential of clinical use.

Description

SARS nucleic acid vaccine and preparation method thereof, and application of related coronavirus S gene in vaccine preparation I. TECHNICAL FIELD

The invention belongs to the field of biological genetic engineering, and particularly relates to an atypical pneumonia (SARS) nucleic acid vaccine and a preparation method thereof, and the application of the SARS-associated coronavirus S gene in preparing a vaccine for preventing SARS. 2. Background Technology

Atypical pneumonia broke out in Guangdong, China, late last year. The disease is acute, highly contagious, and antibiotic treatment has failed. At present, the disease has spread to more than 30 countries and regions, and the WHO has named it severe acute respiratory syndrome (Severe Acute Respiratory Syndrome).

At present, scientists from many countries around the world have independently isolated new-type coronaviruses from different patient sera. Analysis of the viral gene sequence shows that they have 50% to 60% homology with known coronaviruses. The World Health Organization (WHO) has announced that the pathogen of SARS is a mutant strain of coronavirus.

Coronavirus is a non-segmental positive-strand RNA virus with a genome of nearly 30kb that can be transmitted in humans and animals. It mainly infects the respiratory systems of humans and animals. Coronavirus particles are a kind of virus There are four types of structural proteins in viruses: spike (Sike), membrane (Mmbrance, M), envelope (Evelop, E), and nucleoprotein (N). Because coronavirus is an RNA virus, it is very unstable and is susceptible to mutations to avoid host immune surveillance and rejection. Therefore, one must look for The SARS-associated coronavirus has a stable and immunoprotective antigen for the development of related vaccines.

Currently, inactivated virus particle vaccines are rarely used because whole virus particles carry the entire genome of the virus and safety concerns are greater. Although previous coronaviruses are easily obtained in vitro, SARS-associated coronaviruses are extremely toxic and their genetic background is not completely clear, so large-scale preparation of coronavirus particles is not feasible. The development of genetic engineering technology has provided great convenience for the development of subunit vaccines, and the operability and biological safety of subunit vaccines are good. If an immunogenic determinant can be screened and combined with genetic engineering technology, the epitope can be easily modified to enhance its stability, immunogenicity, and biological safety. Obviously, with the help of genetic engineering technology, it is very convenient to prepare effective subunit vaccines.

According to current research, among the four structural proteins known to the coronavirus, spike S is a structural protein that induces a protective immune response. Some scholars have confirmed that the C-terminus of the spike protein of coronavirus is Its epitope is located. At the same time, SARS-associated coronavirus causes infection through the respiratory tract, and there is no vaccine to prevent SARS. Third, the content of the invention

The first object of the present invention is to provide a nucleic acid vaccine capable of preventing the epidemic disease atypical pneumonia (SARS), to better prevent the occurrence and spread of "atypical pneumonia"; another object of the present invention is to provide the above-mentioned SARS nucleic acid Method of vaccine and revealing application of SARS-associated coronavirus S gene in vaccine preparation. . The purpose of the present invention is achieved by: extracting the SARS-associated coronavirus S gene (the coronavirus has four structural genes, the S gene is one of which is described in detail below) through biological engineering means, and cloning it into eukaryotic cells An expression plasmid (such as pcDNA3) is prepared through a series of steps such as screening and purification, and finally made into a SARS nucleic acid injection preparation (vaccine).

In the present invention, clones are obtained by cloning the S gene, one of the structural genes of SARS-associated coronavirus, to the pcDNA3 plasmid, and a nucleic acid vaccine is prepared by using the clone. The pcDNA3 plasmid was purchased from Invitrogen.

The Spike gene fragment sequence used is:

Using the S gene sequence published in the Gene bank as a template, design PCR primers based on the sequence as follows:

Primer D 1 5,-GTTG ^ 7 CAACAACTAAACGAACATG-3 ''

Primer D2 5, -GAG ^ r7UGTTCGTTTATGTGTAATG-3 '

Primer D3 5'-TTG ^ rrCACC ^ rGGGAGCTGAGCATGTCGA-3, Primer D4 5, -ATa4 r7UAGA7 ¾ AACAGGCATTACTTCTGT-3 'Full-length amplification primers: Dl and D2

N-terminal amplification primers: Dl and D4

C-terminal amplification primers: D2 and D3

D14 (N-terminus of S gene amplified by D1 and D4 as primers) amplified fragment length: 2172bp D23 (C-terminus of S gene amplified by D2 and D3 as primers) amplified fragment length: 1885 bp SARS nucleic acid vaccine preparation: '

First, collect and isolate the sera of the recovered patients, and then isolate and extract the total coronavirus. The S gene was obtained by reverse transcription, sequencing, and selection. Then, the S gene was cloned into pcDNA3 to obtain a clone (preservation unit: China Type Culture Collection; preservation date: May 18, 2003; deposit number: CCTCC M 203038 E. eoli JM109 / pcDNA3D14). Purification, preparation and other steps to obtain a SARS nucleic acid vaccine. The main technical route is shown in Figure 3.

The present invention is a nucleic acid vaccine, specifically by extracting the SARS-associated coronavirus S gene, that is, the gene in which the SARS-associated coronavirus epitope belongs, and using genetic engineering technology to make it into a nucleic acid vaccine. Compared with the traditional inactivated virus particle vaccine, the invention has high safety and good immunogenicity.

At present, SARS is spreading rapidly around the world. As a viral infectious disease, no effective medicine can be found at present. In this case, prevention is the best method. It has been confirmed that the C-terminus of SARS-associated coronavirus spike protein belongs to its epitope. Therefore, the present invention is based on this finding. The SARS-associated coronavirus spike protein is extracted and cloned into pcDNA3, which is amplified, purified, and formulated to effectively induce the body to produce antibodies and prevent the virus from infecting the body. Clinical application prospects.

4. Description of the Drawings

Figure 1 is the primer design for DNA vaccine and protein subunit vaccine research;

Figure 2 is an electrophoresis diagram of pcDNA3 plasmid identification of S gene transformation.

Figure 3 is a technical roadmap for the preparation of a SARS nucleic acid vaccine.

V. Specific implementation

The present invention will be further described below through specific examples. Example 1

Preparation of SARS nucleic acid vaccine:

After obtaining the SARS-associated coronavirus sPike (S _F , S _N , S _M , S _c ) gene, the PCR method was used to amplify it. After PCR, it was digested with EcoRl and digested with pcDNA3, and ligated to transform E. coli. Positive clones were screened for ampicillin (Amp resistance), and the SARS nucleic acid vaccine was obtained by culturing, purifying, and formulating.

The dosage form is an injection.

The SARS vaccine contains the SARS-associated coronavirus S gene and the pcDNA3 plasmid. Cloning to PcDNA3 is the full length of the SARS-associated coronavirus S gene.

The cloned into the eukaryotic expression plasmid was the Si region of the S gene of the SARS-associated coronavirus.

A fragment of the S ₂ region of the S gene of the SARS-associated coronavirus was cloned into the eukaryotic expression plasmid.

The cloned into the eukaryotic expression plasmid was the SARS-associated coronavirus S gene region fragment (base number: 49 ~ 3585, see attached table 1).

The cloned into the eukaryotic expression plasmid were the transmembrane segment of SARS-associated coronavirus S gene (base numbers: 3686 ~ 3648, see attached table 1) and the C-terminal fragment.

Example 2

The N-terminal fragment of the S gene of SARS-associated coronavirus was cloned into PcDNA3. The rest is the same as in Example 1.

Example 3

The cloned into PcDNA3 was the middle segment of SARS-associated coronavirus S gene. The rest is the same as in Example 1.

Example 4

The C-terminal fragment of the S gene of SARS-associated coronavirus was cloned into PcDNA3. The rest is the same as in Example 1.

Example 5

S gene transformation pcDNA3 plasmid and identification:

The S gene amplified by PCR was digested with EcoRl, and the pcDNA3 plasmid was digested at the same time, ligated, transformed into E. coli, cultured, and the positive clones were screened by ampicillin resistance to obtain the S gene pcDNA3 recombinant, which was subjected to conventional agarose gel electrophoresis The results are shown in Figure 2.

Example 6

SARS nucleic acid vaccine effect test:

Test animals: 15 Visting rats, weighing about 100g (provided by the Animal Center of Sun Yat-sen University).

Test substance: pcDNA3-S _N Shuttle- S _c

Control: pcDNA3 empty vector

experiment method:

Visting rats were randomly divided into 3 groups, 5 in each group. The first group was the pcDNA3-S> test group; the second group was the Shuttle-S _c test group; the third group was the pcDNA3 empty vector. And tail vein injection.

The first group: pcDNA3-S _N test group

Rats No.1 and No.2 were intramuscularly injected with 50 μg pcDNA3-S _{N on} each of their four legs, that is, a total of 200 μg.

In mice 3 and 4, 200 pcDNA3-S _N was injected into the tail vein of each mouse to add a mixed volume of mycoplasma 2000.

No. 5 rats were not injected.

The second group: Shuttle-Sc test group

Rats No. 1 and No. 2 were intramuscularly injected with 25 μg of Shuttle-S _{c on} each of their four legs, that is, a total of 100 μg per mouse.

3,4 mice, each mouse tail vein injection of 25 μ g Shuttle- S _c is added to the mixed volume of 2000 branched plastid.

No. 5 rat was not injected

The third group: pcDNA3 empty vector group

In mice No. 1 and No. 2, each leg of each mouse was intramuscularly injected with 50 μg pcDNA3 empty vector, that is, a total of 100 g per mouse.

In mice 3 and 4, 50 μg pcDNA3 empty vector was injected into the tail vein of each mouse and mixed volumes of mycoplasma 2000 were added.

No. 5 rat was not injected

On the 7th day and the 14th day after the injection, a booster was used at the same dose, and the blood serum was collected on the 21st day.

Expected results: sera of animals injected pcDNA3-S _N and Shuttle- S _c of antibody expression can be detected in the corresponding fragment SARS virus; uninjected animals and the control group could be detected SARS virus antibody.

Results. 'The above experiments were performed three times and all achieved the expected results.

Claims

Claim

1. A SARS vaccine, characterized in that it comprises a SARS-associated coronavirus S gene and a eukaryotic expression plasmid.

2. The vaccine according to claim 1, characterized in that the eukaryotic expression plasmid comprises a CMV promoter and BGHpolyA.

3. The vaccine according to claim 1 or 2, characterized in that the eukaryotic expression plasmid is pcDNA3.

4. The vaccine according to claim 1 or 2, characterized in that the full length of the SARS-associated coronavirus S gene is cloned into the eukaryotic expression plasmid.

5. The vaccine according to claim 1 or 2, characterized in that the cloned into the eukaryotic expression plasmid is a fragment of the Si region of the S gene of the SARS-associated coronavirus.

6. The vaccine according to claim 1 or claim 2, characterized in that the cloning of the SARS-associated coronavirus S gene fragment ₂ S region eukaryotic expression plasmid.

7. The vaccine according to claim 1 or 2, characterized in that the cloned into the eukaryotic expression plasmid is a fragment of the S gene region of the SARS-associated coronavirus.

8. The vaccine according to claim 1 or 2, characterized in that the fragments cloned into the transmembrane region of the S gene of the SARS-associated coronavirus and the C-terminal fragment are cloned into the eukaryotic expression plasmid.

9. A method for preparing a SARS nucleic acid vaccine, comprising:

(1) obtaining the S gene of SARS-associated coronavirus;

(2) clone the S gene into the PcDNA3 plasmid;

(3) After culturing, expanding, and purifying, a preparation is prepared.

10. The method for preparing a nucleic acid vaccine according to claim 9, characterized in that The S gene was taken and amplified by PCR. At the same time, the S gene was digested with PcDNA3 and ligated to transform E. coli. Positive clones were screened using ampicillin (Amp ¹ ") resistance, and cultured and purified to prepare a preparation.

11. The method for preparing a nucleic acid vaccine according to claim 9, wherein the S gene is cloned into the PcDNA3 plasmid and digested with EcoR1.

12. The method for preparing a nucleic acid vaccine according to claim 9, characterized in that the PCR primers are designed according to the S gene sequence as follows:

Primer D1 5 '-GTTG ^^ 7TCAACAACTAAACGAACATG-3'

Primer D2 5, -GA ^ 7TCGTTCGTTTATGTGTAATG-3, Primer D3 5 '-TTGAATTCACCATGGGAGCTGAGCATGTCGA-S' Primer D4 5'-ATG ^ 4rrCAGAr∑4AACAGGCATTACTTCTGT-3 'Full-length amplification primers: Dl and D2

N-terminal amplification primers: Dl and D4

C-terminal amplification primers: D2 and D3

D14 (N1 terminus of S gene amplified by D1 and D4 as primers) Amplified fragment length: 2172bp

D23 (C-terminus of S gene amplified by D2 and D3 as primers) Amplified fragment length: 1885 bp

13. The method for preparing a nucleic acid vaccine according to claim 8 or 9, wherein the preparation is an injection.

14. Application of the SARS-associated coronavirus S gene in the preparation of a vaccine to prevent SARS.