Sastry K S R, Tuteja U, Bala Krishna K, Batra H V
Division of Microbiology, Defence Research & Development Establishment, Gwalior, India.
Indian J Med Res. 2003 Mar;117:111-8.
BACKGROUND & OBJECTIVE: Anthrax has been reported from almost every country and India is endemic for this disease. There is considerable under reporting of the disease because of lack of microbiological facilities and diagnostic reagents. In India only conventional methods which have limitations, are being used to diagnose the disease. Hence the aim of this study was to isolate and purify protective antigen (PA) using different protocols and to use this PA for detection of anti-PA antibodies from sera samples.
Protective antigen was isolated and purified from the Sterne strain of Bacillus anthracis. B. anthracis lacking pXO1 and pXO2 transformed with pYS5 (B. anthracis pYS5) and recombinant Escherichia coli transformed with pQE30 containing PA gene using hydroxyapatite (HA), Q-sepharose fast protein liquid chromatography (FPLC) and nickel-nitrilotriacetic acid (Ni-NTA) chromatographic methods, respectively. A mixture of PA and edema factor (EF) was injected subcutaneously into rabbits to test the biological activity of PA. The immunogenicity of PA was tested by inoculating the protein into rabbits along with adjuvant. Using this PA, 20 bovine sera samples (pre- and post-vaccinated) were tested by Western blotting (WB) for the presence of anti-PA antibodies.
The 83 kDa PA protein was obtained from all the bacteria with the yields of 13, 50 and 9.0 mg/l from Sterne B. anthracis, B. anthracis pYS5 and recombinant Esch. coli, respectively. Formation of edematous ulcers at the site of PA+EF injection clearly confirmed the retention of biological activity of the proteins. Of the 10 post-vaccination sera tested, 9 showed clear positive by WB whereas none of the pre-vaccination sera showed the reaction.
INTERPRETATION & CONCLUSION: The purified PA preparations obtained in the present study may possibly be utilized for detection of anti-PA antibodies in the sera of anthrax patients for timely diagnosis of the disease and, might also be tested for their efficacy and use as human anthrax vaccine.
几乎每个国家都报告过炭疽病例，印度是这种疾病的地方性流行区。由于缺乏微生物学设施和诊断试剂，该病的报告存在大量漏报情况。在印度，仅使用有局限性的传统方法来诊断该疾病。因此，本研究的目的是使用不同方案分离和纯化保护性抗原（PA），并使用该PA检测血清样本中的抗PA抗体。
从炭疽芽孢杆菌的Sterne菌株中分离和纯化保护性抗原。分别使用羟基磷灰石（HA）、Q-琼脂糖快速蛋白质液相色谱（FPLC）和镍-次氮基三乙酸（Ni-NTA）色谱方法，对缺乏pXO1和pXO2并经pYS5转化的炭疽芽孢杆菌（炭疽芽孢杆菌pYS5）以及经含有PA基因的pQE30转化的重组大肠杆菌进行处理。将PA和水肿因子（EF）的混合物皮下注射到兔子体内，以测试PA的生物活性。通过将该蛋白质与佐剂一起接种到兔子体内来测试PA的免疫原性。使用该PA，通过蛋白质印迹法（WB）检测20份牛血清样本（接种疫苗前后）中抗PA抗体的存在情况。
从所有细菌中均获得了83 kDa的PA蛋白，来自炭疽芽孢杆菌Sterne菌株、炭疽芽孢杆菌pYS5和重组大肠杆菌的产量分别为13、50和9.0 mg/l。在PA + EF注射部位形成水肿性溃疡，清楚地证实了蛋白质的生物活性得以保留。在测试的10份接种疫苗后的血清中，9份通过WB显示为明显阳性，而接种疫苗前的血清均未显示出反应。
本研究中获得的纯化PA制剂可能可用于检测炭疽患者血清中的抗PA抗体，以便及时诊断该疾病，并且还可能对其作为人类炭疽疫苗的功效和用途进行测试。
