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Oral vaccine of streptococcus agalactiae and preparation method thereof

A technology for streptococcus nisca and oral vaccine, which is applied in the field of oral vaccine, can solve the problems of difficulty in playing a protective role, large fish stress response, weak immune effect, etc., and achieves convenient administration, small immune stress response, and operation. simple effect

Active Publication Date: 2014-06-04
TONGWEI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally speaking, intraperitoneal or intramuscular injection is easy to control the dose, and the immune response is better, but the stress response of the fish body is large, and the wound is prone to secondary infection
The operation of oral immunization is simple, and the stress response of the fish body is small. However, the immune effect of the existing Streptococcus agalactiae vaccine is very weak and it is difficult to play a protective role.

Method used

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  • Oral vaccine of streptococcus agalactiae and preparation method thereof
  • Oral vaccine of streptococcus agalactiae and preparation method thereof
  • Oral vaccine of streptococcus agalactiae and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 Construction of oral vaccine of the present invention

[0037] 1. Cloning of target gene fragments

[0038] 1.1 Method

[0039] Streptococcus agalactiae was cultured to the exponential phase with BHI broth, and the bacterial cells were collected by centrifugation using a bacterial DNA extraction kit to collect and extract bacterial DNA of Streptococcus agalactiae, and a large number of sip genes were obtained by PCR amplification.

[0040] The upstream and downstream primers of Sip gene are

[0041] sip upstream primer: 5'- GGATCC CTGCCACTTCAATGAAA-3' (the underline is the added BamH Ⅰ restriction site, and the bold is the Kozak sequence)

[0042] sip downstream primer: 5'- AAGCTT TTATTTGTTAAATGATACGTG-3' (underlined is the added HindⅢ restriction site)

[0043] PCR reaction system: 1 μl of upstream and downstream primers; Mix12.5 μl; DNA template 1 μl; ddH 2 O9.5 μl. Reaction conditions: 95°C pre-denaturation for 5 minutes, 94°C for 50s, 55°C fo...

Embodiment 2

[0079] Stability and safety detection of embodiment 2 oral vaccine of the present invention

[0080] SL7207, SL7207 (pVAX1) and SL7207 (pVAX1-sip) were prepared according to the method in Example 1.

[0081] 1. Detection method

[0082] (1) Growth characteristics test of recombinant bacteria

[0083] SL7207, SL7207 (pVAX1) and SL7207 (pVAX1-sip) were streaked and inoculated in LB solid medium with an inoculation loop, cultured at 37°C for 24 hours, and single colonies were picked and inoculated in LB liquid medium respectively, 37 Cultivate with shaking at 120r / min overnight at 120°C, take 1 mL of the bacterial liquid into a flask containing 100 mL of LB medium the next day, and culture with vigorous shaking at 37°C, measure the OD of the bacterial liquid every 1 h 600 Value once, measure 18h in total, and draw the growth curve of the measured bacteria.

[0084] (2) In vivo stability test of SL7207 (pVAX1-sip)

[0085] Divide 40 healthy tilapias of about 100g randomly into...

Embodiment 3

[0107] Embodiment 3 Effectiveness detection of oral vaccine of the present invention

[0108] In this chapter, the oral immunity effect of vaccine on tilapia was investigated by two inoculation methods of direct gavage and mixing. Gastric inoculation can quantitatively examine the impact of immunization dose on the immune effect under laboratory conditions. The mixing method focuses on simulating the actual use of production methods to evaluate the immune effect of the vaccine. The results obtained are more reference for later field trials or production promotion and application value. In this chapter, two indicators of specific antibody titer and immune protection rate were used to evaluate the immune effect of the vaccine on tilapia.

[0109] 1 material

[0110] 1.1 Test animals

[0111] Healthy tilapia: 900 healthy tilapia weighing about 100g, purchased from Tongwei tilapia breeding base, and used after a week of temporary breeding.

[0112] 1.2 Strains

[0113] Strept...

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Abstract

The invention discloses a nucleotide sequence as shown in SEQ ID No.: 1, and a recombinant vector and recombinant bacteria containing the nucleotide sequence. The invention also discloses oral vaccine of streptococcus agalactiae. The oral vaccine of streptococcus agalactiae is characterized by being grafted and immunized by an oral manner and has the advantages of being high in protection rate for tilapia mossambica, convenient to administrate, simple in operation, small in immunological stress reaction, safe, and high in application prospect.

Description

technical field [0001] The invention relates to an oral vaccine for Streptococcus agalactiae. Background technique [0002] Streptococcus agalactiae, also known as group B streptococci (GBS), belongs to the family Streptococcus (Streptococcaceae), Streptococcus (Streptococcus), is a facultative Gram-positive coccus, according to The antigenicity of the capsular polysaccharides can be divided into 10 serotypes (types Ia, Ib, II-VIII), which are the main pathogenic bacteria causing Streptococcus tilapia. [0003] In recent years, large-scale tilapia streptococcal disease outbreaks have occurred in major tilapia production areas such as Guangdong, Hainan, and Fujian, which have spread widely and are difficult to control, causing huge economic losses to farmers. The diseased fish showed poor spirit and appetite, black body color, wandering around in circles, and some diseased fish had lens opacity, exophthalmos and even loss of eyes; Positive correlation, when the temperature ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/79C12N1/21A61K39/09A61P31/04C12R1/42
Inventor 刘天强肖丹汪开毓黄凌远黄冠军阳涛
Owner TONGWEI
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