2021年3月，山東省龍口市某養豬場發(店服fā)生了一起(qǐ)以敗血症爲主要臨床症狀的烈性傳染藍光病。爲對(duì)引起(qǐ)此次疫病的病原進(jìn)行确診，無菌采集發(問吃fā)病死亡仔豬的肝髒、脾髒等髒器進(jìn)行染色鏡檢、病原分地用離培養、生化鑒定及PCR檢測。結果顯示，本次分離株的形态結慢東構、培養特性及生化結果與多殺性巴氏杆菌較爲一緻，且可特異性擴增出多殺性巴自她氏杆菌特異性基因Kmt，因此本次山遠分離菌株爲一株豬源多殺性巴氏杆菌，命名爲LK01開動。随後(hòu)，對(duì)該分離株的5個主要抗原雪師蛋白基因（OmpH、OmpA、AspA、pIpE和TA很短A）進(jìn)行克隆與原核表達。先將(jiāng道拿)PCR成(chéng)功擴增出的目的片段與雙酶切回收後(hòu)的p志哥Cold-I載體進(jìn)行同源重組連接，測序結果正确的重組質些還粒轉化至大腸杆菌BL21（DE3）感受為街态細胞中，随後(hòu)經(jīng)IPTG誘導後(hòu)進(jìn)行W舊業estern blot檢測。結果麗如顯示，在30.0、21.5、52.0、37.5和習美45.5 kDa處出現特異性條帶，表明各重組蛋白均成(chéng)功表達。刀刀本研究爲開(kāi)展豬源多殺性巴氏杆菌懂務病的流行病學(xué)研究，開(kāi)發(fā)新型重組疫苗與診斷試劑奠定紙錢了基礎。
Isolation and Identificat門員ion of Porcine Pasteu暗可rella multocida Strain and Prokar男長yotic Expression of房綠 Its Major Antigen Proteins
In March 2021，a se錢如vere infectious disease with major symp畫日tom of septicemia occurred in兵一 a swine farm in Longkou Ci算美ty，Shandong Province. In order to ident來就ify the pathogen le輛和ading to the disea睡工se，livers and spleens of dead pigle醫低ts were aseptically col要票lected for staining microscopic exa答她mination，bacteri畫市al isolation an光購d culture，biochemical iden我務tification and PCR detection. T多錢he results showed that子個 the morphological structure，cult做自ure characteristi她民cs and the results of bioc家習hemical test of t算體he isolate were relatively c劇亮onsistent with those of Pasteurella mul光著tocida（PM），then Kmt，the specific ge農快ne of PM was succes兵對sfully amplified，it was identified t河算hat the isolate was a porci木男ne PM named as LK01. Su門用bsequently，its fi可也ve major antigen genes（in和空cluding OmpH，OmpA，AspA，pIpE and TA快鐘A）were cloned and 窗拍expressed in prokaryotic e從可xpression system. By homologous家章 recombination，the targe你輛t fragments amplifi離中ed by PCR were link務麗ed with the pCold-I城信 that was after double digestion.海歌 The recombinant pl器子asmid with correct sequen慢資cing results was transform制懂ed into E. coli BL21（DE3）cell中志s and induced by IPTG fo上費r Western blot detection. It was兒房 observed that specific b筆著ands appeared at 30.0，21.5，5錯中2.0，37.5 and 45.5問木 kDa，indicating 老件that all recombinant proteins were s如信uccessfully expressed. A founda體男tion was thus provided for fu文也rther epidemiolo船錢gical study on porcine PM and fo來綠r development of new recombinant vaccin哥動es and diagnostic reagents.
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