中文摘要：

目的：构建表达猪卵透明带蛋白（pZP3α）的重组乳酸杆菌。方法：用PCR方法扩增得到短小乳酸杆菌染色体中的S-层蛋白的信号肽序列和猪卵透明带的pZP3α基因,并依次克隆到乳酸杆菌整合型表达质粒pIlac的lac启动子下游,得到质粒pIlac-pZP3α。将质粒pIlac-pZP3α电穿孔转化干酪乳酸杆菌（L.casei CECT5276）,挑选转化后的耐药菌落,在含5μg/mL红霉素的MRS培养基中培养,抽提其基因组DNA做模板,PCR鉴定验证pZP3α基因是否整合到乳酸杆菌的基因组中。筛选重组菌在无红霉素选择压力下传代培养直至发生第2次重组使红霉素抗性基因消失。结果：酶切与测序结果表明信号肽和pZP3α基因正确克隆到载体pIlac中;PCR鉴定证实pZP3α基因成功重组到乳酸杆菌的基因组中,培养50 d后重组乳酸杆菌抗性消失,用斑点免疫印迹化学发光法检测到经终质量分数为0.75%乳糖诱导后的上清中有pZP3α蛋白分泌。结论：成功构建表达pZP3α的重组乳酸杆菌,pZP3α在乳酸杆菌中得到了稳定的、分泌表达。

英文摘要：

Aim： To constructa recombinant lactbacillus strain excreting porcine zona pellucida-3α（pZP3α）. Methods： The signal peptide sequence of S-layer protein from L. brevis cloned into downstream of lactse-inducible promoter of an integrative plasmid pllac, and then ligated the pZP3α sequence into down-stream of S-layer protein to get plasmid pllac-pZP3α. After electoporation into L. casei CECT 5276, PCR using the genomic DNA of the recombinant lactbacillus as template was performed to confirm whether the pZP3α gene had been integrated into the genome. The recombinant lactbacillus grown in MRS broth without antibiotic in order to allow the second recombination event, which would excise the antibiotic gene. Results： Restricton endonuclease and sequence analysis verified that the fragment cloned was correct The pZP3α were integrated into the genome of L. casei CECT 5276 which proved by PCR. After grow in MRS 50 days the antibiotic gene have been excised. Chemiluminescence Immunoassay （ CLIA ） indicate that the concentration ofpZP3α in the culture after lactse inducton 24 h was 0. 75%.Conclusion：We have got stable and efficient pZP3α excretion in L. casei that were inducible by lactose.