【摘要】：為探究尼羅羅非魚無乳鏈球菌(GBS)莢膜多糖合成基因cpsE和neuA對菌株生物學(xué)特性的影響,本研究利用同源重組的方法,構(gòu)建了GBS的cpsE與neuA的單基因缺失突變株。具體方法為:用Infusion-PCR的方法分別構(gòu)建帶有氯霉素抗性基因的cpsE與neuA基因敲除重組質(zhì)粒pSET4s-cpsE和pSET4s-neuA。將構(gòu)建好的質(zhì)粒電轉(zhuǎn)化入GBS感受態(tài)細(xì)胞中,通過改變培養(yǎng)溫度實(shí)現(xiàn)雙交換和質(zhì)粒丟失,最后經(jīng)氯霉素抗性篩選獲得疑似敲除株。通過菌落PCR、RT-PCR及DNA測序等方法對疑似敲除株進(jìn)行驗(yàn)證。結(jié)果顯示GBS的兩個(gè)突變株ΔcpsE和ΔneuA被成功構(gòu)建。在此基礎(chǔ)上,通過生物學(xué)功能分析比較基因缺失突變株ΔcpsE、ΔneuA與野生株在菌株生長速率、莢膜多糖厚度、唾液酸含量和毒力方面的差異。結(jié)果發(fā)現(xiàn)缺失突變株ΔcpsE和ΔneuA的生長速度與野生株無顯著差異,但莢膜多糖厚度、唾液酸含量和菌株毒力均顯著低于野生株。進(jìn)一步研究顯示,cpsE是魚源GBS莢膜多糖合成的關(guān)鍵基因,neuA基因則是莢膜多糖唾液酸化的關(guān)鍵基因,它們的缺失導(dǎo)致了GBS莢膜唾液酸含量的降低,且顯著降低了菌株的毒力。
[Abstract]:In order to investigate the effects of cpsE and neuA on the biological characteristics of Streptococcus niloticus (GBS) capsule polysaccharides synthesis genes, homologous recombination was used to construct the single gene deletion mutant of cpsE and neuA of GBS. The specific methods are as follows: construction of cpsE and neuA gene knockout recombinant plasmids pSET4s-cpsE and pSET4s-neuA. with chloramphenicol resistance gene by Infusion-PCR The constructed plasmids were electrically transformed into GBS receptive cells, and the double exchange and plasmid loss were realized by changing the culture temperature. Finally, the suspected knockout strain was obtained by chloramphenicol resistance screening. The suspected knockout strain was verified by colony PCR,RT-PCR and DNA sequencing. The results showed that two GBS mutants 螖 cpsE and 螖 neuA were successfully constructed. On the basis of biological function analysis, the difference of growth rate, capsule polysaccharide thickness, sialic acid content and virulence between the mutant 螖 cpsE, 螖 neuA and wild strain was compared. The results showed that the growth rate of 螖 cpsE and 螖 neuA was not significantly different from that of wild strain, but the thickness of capsule polysaccharide, the content of sialic acid and virulence of strain were significantly lower than that of wild strain. Further studies showed that cpsE was a key gene for the synthesis of GBS capsule polysaccharides from fish, while neuA gene was a key gene for sialic acid acidification of capsule polysaccharides. Their deletion resulted in a decrease in the content of sialic acid in the capsule of GBS and significantly reduced the virulence of the strain.
【作者單位】： 中國水產(chǎn)科學(xué)研究院珠江水產(chǎn)研究所農(nóng)業(yè)部熱帶亞熱帶水產(chǎn)資源利用與養(yǎng)殖重點(diǎn)實(shí)驗(yàn)室;上海海洋大學(xué)水產(chǎn)與生命學(xué)院;
【基金】：國家自然科學(xué)基金項(xiàng)目(NSFC)(31272688) 現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)資金項(xiàng)目(CARS-48) 中國水產(chǎn)科學(xué)研究院中央級公益性科研院所基本科研業(yè)務(wù)費(fèi)專項(xiàng)資金項(xiàng)目(2017YH-ZC06)
【分類號】：S943

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