CAJ | 학술논문

LrrG和表面免疫原性蛋白（ Sip）是无乳链球菌（ Streptococcus agalactiae）的2种表面蛋白，具有良好的免疫原性。为获得罗非鱼无乳链球菌表面蛋白LrrG和Sip蛋白的融合蛋白，该试验采用基因拼接技术中的双酶切法分2步逐个将Sip和LrrG基因插入pCold Ⅱ载体中，构建原核表达载体pCold Ⅱ-LrrG-Sip。将成功构建的融合基因原核表达载体转化感受态细胞BL21（DE3），进行诱导表达条件的优化。结果显示，15℃、IPTG 0.5 mmol·L-1诱导9 h，目的蛋白呈可溶状态的表达量最高。Western Blot检测结果显示LrrG-Sip融合蛋白大小与预测一致（162 kDa），说明成功构建了融合基因，为罗非鱼源无乳链球菌亚单位疫苗的研制奠定了基础。
LrrG화표면면역원성단백（ Sip）시무유련구균（ Streptococcus agalactiae）적2충표면단백，구유량호적면역원성。위획득라비어무유련구균표면단백LrrG화Sip단백적융합단백，해시험채용기인병접기술중적쌍매절법분2보축개장Sip화LrrG기인삽입pCold Ⅱ재체중，구건원핵표체재체pCold Ⅱ-LrrG-Sip。장성공구건적융합기인원핵표체재체전화감수태세포BL21（DE3），진행유도표체조건적우화。결과현시，15℃、IPTG 0.5 mmol·L-1유도9 h，목적단백정가용상태적표체량최고。Western Blot검측결과현시LrrG-Sip융합단백대소여예측일치（162 kDa），설명성공구건료융합기인，위라비어원무유련구균아단위역묘적연제전정료기출。
LrrG( leucine-rich repeat protein from GBS)and Sip( surface immunogenic protein),which are two kinds of surface anti-gen proteins from Streptococcus agalactiae in tilapia,have good immunogenicity. To obtain the LrrG-Sip fusion protein via coalescing surface antigen protein LrrG and Sip of S. agalctiae in tilapia,we cloned Sip and LrrG genes into vector pCold Ⅱ one by one using double enzyme method of gene splicing technology,and constructed a prokaryotic expression vector pCold Ⅱ-LrrG-Sip. The recombi-nant plasmid was transformed into E. coli BL21(DE3),and the result indicated that 9 h,15 ℃,0. 5 mmol·L-1IPTG were the opti-mum inducing conditions under which fusion protein was most soluble and abundant. Western blotting test showed that the LrrG-Sip fu-sion protein was about 160 kDa,consistent with the prediction(162 kDa),which suggested the prokaryotic expression vector pColdⅡ-LrrG-Sip was constructed successfully and laid the foundation for developing subunit vaccines for S. agalctiae in tilapia.