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Streptococcus protective antigen SAP and preparation method thereof

A protective antigen, streptococcus technology, applied in biochemical equipment and methods, chemical equipment and methods, viruses/bacteriophages, etc., can solve problems such as difficult to effectively control SEZ infection, and achieve high protective efficacy and strong specificity Effect

Inactive Publication Date: 2016-10-12
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Effective control of SEZ infections has been difficult due to a lack of comprehensive understanding of SEZ virulence factors and protective antigens
Currently, no effective antigens are available for the development of vaccines against SEZ

Method used

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  • Streptococcus protective antigen SAP and preparation method thereof
  • Streptococcus protective antigen SAP and preparation method thereof
  • Streptococcus protective antigen SAP and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0029] The preparation method of the above-mentioned streptococcal protective antigen SAP is as follows:

[0030] 1) PCR amplification: use the SEZ MGCS10565 genome (NCBI Accession: CP001129.1) as a template, and use primers for PCR amplification. The primers are:

[0031] Forward primer (SEQ ID NO.3): 5′-CATGCG GGATCC CAAGTA -3′, the underlined part is BamHI Restriction sites;

[0032] Reverse primer (SEQ ID NO.4): 5′-AATCTTCTGGTCGACACTT-3′, the underlined part is Sal I Restriction sites.

[0033] 2) Ligation with the vector: the PCR product was digested with a restriction endonuclease, and the recovered PCR digest product was ligated with the pET-32a vector to obtain a recombinant vector.

[0034] 3) Transformation and induction: After transforming E. coli with the recombinant vector, incubate until the exponential growth phase, and continue to incubate for 3 hours after adding IPTG.

[0035] Preferably, the Escherichia coli is BL21 competent cells; the added IPTG i...

Embodiment 1

[0042] Example 1 Determination of antibody titer

[0043] Serum IgG titer was measured by ELISA, and the purified recombinant protein SAP was coated on the enzyme-linked plate, and the blood was collected from the mice 10 days after the second immunization to separate the serum. After serial dilution, 100 μL of each dilution was added to the enzyme-linked plate. After reacting at 37°C, add rabbit anti-mouse IgG, read the OD value with a microplate reader, and take the maximum dilution factor of the serum whose OD value is greater than the average OD value of the control serum + 3 times the variance SD (standard deviation) as the serum antibody. In order to infer the immune type, IgG1 and IgG2a were further used as controls to measure Th2 and Th1 immune responses, respectively.

[0044] Experimental results: the IgG titer level of the immune group was significantly higher than the antibody level of the negative control group (p figure 1 A).

[0045]The results showed that SAP...

Embodiment 2

[0046] Example 2 Mice immunization and challenge test

[0047] 1. Forty 4-week-old BALB / c female mice were randomly divided into 4 groups, 10 in each group;

[0048] 2. Experimental group: After 50 μg of purified recombinant SAP protein was emulsified with Marcol 52 adjuvant, the mice in the first group were immunized by intraperitoneal injection, and the mice were immunized for the second time in the same way 14 days later;

[0049] 3. Positive control group: After 50 μg of purified recombinant SzP protein was emulsified with Marcol 52 adjuvant, the mice in group 1 were immunized by intraperitoneal injection, and immunized mice were injected for the second time in the same way 14 days later;

[0050] 4. Negative control group: inject PBS emulsified with Marcol 52 adjuvant to mice in group 3;

[0051] 5. Blank control group: inject PBS to the mice in group 4;

[0052] 6. Ten days after the second immunization injection of all mice, the blood was collected from the tail vein ...

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Abstract

The invention provides streptococcus protective antigen SAP and a preparation method thereof. The antigen SAP is SAP recombination protein of SEZ. The preparation method comprises the following steps: firstly obtaining a target gene through a PCR technology, connecting the target gene to a carrier and converting escherichia coli, and performing purification on an expression product induced by escherichia coli, so as to finally obtain target recombination protein. The streptococcus protective antigen SAP has high conservatism, vaccine prepared from the protein is high in specificity, a series of bioengineering technologies and mouse experimental analysis prove that the SAP can provide higher protective efficacy after immunization, anti-SAP mice second immune serum has obvious passive immune protection to mice, and high-level antibody titer is expressed in mice serum of the SAP after immunization. The transcriptional level of SAP gene in mice body infected by SEZ is higher than that of SAP gene cultured in vitro, and an SAP antibody can induce high-level bactericidal ability.

Description

technical field [0001] The invention relates to the field of biological products, in particular to a Streptococcus protective antigen SAP and a preparation method thereof. Background technique [0002] Streptococcus suis is a bacterial disease that endangers the pig industry today, seriously endangering the development of the pig industry in my country and even in the world. The researchers determined that the isolation rate of Streptococcus suis was the highest through the isolation and identification of bacteria from 2,912 samples of porcine respiratory diseases in China, indicating that Streptococcus suis is the first bacterial disease in porcine in my country. Streptococcus suis not only seriously endangers the development of animal husbandry, but also causes public health problems that cannot be ignored on a global scale. The pathogens of Streptococcus suis in my country mainly include Streptococcus suis type 2 (SS2, Streptococcus suis ) and Streptococcus equi subspec...

Claims

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Application Information

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IPC IPC(8): C07K14/315C12N15/70
CPCC07K14/315C12N15/70C12N2800/101
Inventor 付强魏子贡马春全王爱富李桂珍罗惠娜
Owner FOSHAN UNIVERSITY
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