A systematic analysis of the effects of fine-structures of antibodies on microbial expression and secretion and construction of a general-purpose system for antibody production.

(1)构建了含73个氨基酸残基和83个氨基酸残基的单链Fab基因，并在短芽孢杆菌中进行了表达。(2)为构建短小芽孢杆菌产生抗体片段的新型表达分泌载体，对短小芽孢杆菌隐蔽表达载体pWT481的ORFs进行了功能分析。发现ORF(ORF3)的缺失降低了细菌在生长稳定期的存活率。(3)编码CH1结构域的基因被引入不同的缺失。将获得的基因片段直接与地衣芽孢杆菌α-淀粉酶基因的5‘端融合，并在短芽孢杆菌中表达。用BLA抗血清进行Western印迹分析，结果表明CH1结构域N端的4个氨基酸残基对CH1结构域的表达和/或分泌具有抑制作用。

(1) Two genes coding for single-chain Fabs containing a linker peptide of 73 and 83 amino acid residues, respectively, were constructed and then expressed in Bacillus brevis. Their products were accumulated in the medium at a level of 200 mg/l.(2) To construct a new expression-secretion vector for production of antibody fragments by B.brevis, functional analysis of ORFs of pWT481, a cryptic plasmid in B.brevis 481, was carried out. Deletion of an ORF (ORF3) was found to decrease the survival rate of the bacterium at the stationary phase of growth.(3) Various deletions were introduced into the cDNA encoding CH1 domain. The resultant cDNAs were directly fused to the 5' terminus of the Bacillus licheniformis α-amylase (BLA) gene and then expressed in B.brevis. Amounts of the products of the fused genes accumulated in the medium were determined by Western blot analysis by using an antiserum against BLA.The results suggested that the 4 amino acid residues at the N-terminus of CH1 domain are inhibitory for the expression and/or secretion of CH1 domain.