Streptococcus suis 2 secrete nuclease SsnA genes and preparation method and application thereof

[0046] Example 1: Cloning of the expression sequence of Streptococcus suis type 2 secreted nuclease SsnA

[0047] 1) Cultivation of Streptococcus suis type 2 (SS2)

[0048] The Streptococcus suis type 2 strain CVCC606 (purchased from Beijing, China, China Veterinary Drug Administration) was inoculated on TSA solid medium (purchased from Sigma) containing 10% inactivated newborn calf serum, and a single colony was selected for inoculation In TSB liquid medium (purchased from Sigma), place in a shaker (150r / min), shake culture overnight at 37°C.

[0049] 2) SS2 genomic DNA extraction and target gene amplification

[0050] According to the TIANGEN bacterial genome extraction kit (purchased from Wuhan Dafeng Biotechnology Co., Ltd.), the genomic DNA of Streptococcus suis type 2 was extracted according to the operation method provided in the instruction manual of the kit. The primer design software Primer 5.0 was used to design the 377 amino acid sequence used to amplify the secretory nucl

[0068] Example 2: Construction of recombinant expression plasmid pET28a-SsnA

[0069] The PCR product and vector pET-28a(+) (purchased from Noagen) of the target gene SsnA were digested with BamHI and XhoI, respectively, and the digested SsnA fragment and vector pET-28a(+) were recovered; then ligated with T4DNA ligase, In a 16°C water bath overnight, transform DH5a competent cells, culture them on LB solid medium at 37°C, randomly select a number of single colonies from them, put them into LB liquid medium at 37°C for 12 hours, extract plasmids from them, and digest them with enzymes After identification, the positive recombinant plasmid was screened and named pET28a-SsnA. The plasmid map of pET-28a(+) is as follows figure 2 As shown, the recombinant plasmid pET28a-SsnA double restriction digestion map is shown in Figure 5 .

[0070] Description of the above reagents: Taq enzyme and 10×Taq enzyme Buffer, BamH I, Xhol I and other endonucleases and related buffers, T4 ligase and 10×

[0072] Example 3: Preparation of recombinant E. coli BL21 / PET28a-SsnA

[0073] The recombinant expression vector pET-28aSsnA was transformed into E. coli BL21(DE3) competent cells (purchased from Wuhan Life Technology Co., Ltd.), coated with LB kanamycin (kna) (purchased from Invitrogen) plates, and selected The colonies were cultured in LB liquid medium at 37°C, PCR and restriction enzyme digestion were performed, and the correctly identified colonies were expressed with isopropylthio-β-D-galactoside (IPTG) (purchased from Invitrogen) From it, a recombinant Escherichia coli BL21 / PET28a-SsnA that can induce the expression of secreted nuclease SsnA protein in E. coli BL21 was screened.