【研究目的及意义】自转运蛋白(autotransporter，AT）分泌系统是革兰氏阴性菌中病原蛋白跨外膜分泌的重要手段之一，其可通过两个连续的步骤介导蛋白质穿过阴性菌的内外膜，将蛋白质释放到胞外环境或“展示”在细菌表面，这一特性使其可用于目标蛋白在细菌表面的展示，以开发特定生物工程产品。其中，基于自转运蛋白Ag43构建的细菌表面展示系统被认为是展示外源蛋白的理想系统。然而在不同的研究中，外源蛋白取代Ag43乘客结构域时选用的替换点不尽相同，在构建融合蛋白时，是否保留乘客结构域以及需要保留多少Ag43乘客结构域氨基酸才能实现表面展示的最优化，目前还有待研究。因此本研究通过构建含有不同乘客结构域的Ag43表面展示系统，以HPV16L1为外源蛋白，来探索其展示外源蛋白所需的最优化组合。在最优化组合的基础上，利用绿色荧光蛋白（EGFP）、绿色荧光蛋白纳米抗体（nanobody, NB）以及β-葡萄糖苷酶（β-glucosidase，βglC）三种不同的外源蛋白来检测该表面展示载体的功能性表达及通用性。

【研究方法】（1）利用基因工程技术构建Ag43的四种表面展示载体：Ag43/138、Ag43/551、Ag43/552和Ag43/700；（2）利用基因工程技术构建外源蛋白-Ag43融合蛋白表达载体（3）IPTG诱导重组蛋白表达及SDS-PAGE分析；（4）利用胰蛋白酶消化验证外源蛋白在大肠杆菌的表面展示。（5）EGFP、NB及βglC表面展示及活性检测。

【结果】（1）成功构建四种表面展示载体和四种HPV16L1-Ag43重组蛋白表达载体。经IPTG诱导后，构建的四种表面展示载体均能表达HPV16L1蛋白。胰蛋白酶消化后，重组蛋白条带均减弱，其中，Ag43/700-HPV16L1条带减弱最明显。（2）以Ag43/700为表面展示载体，成功构建EGFP-Ag43/700、NB-Ag43/700以及βglC-Ag43/700重组蛋白质粒。经IPTG诱导后，构建的3种重组蛋白均能表达。胰蛋白酶消化后，3种重组蛋白条带均减弱。研究发现：①EGFP-Ag43重组菌在荧光显微镜下可观察到荧光，但未加IPTG组未见荧光；②表面展示的NB可识别结合EGFP，在激光照射下可产生荧光，未加IPTG及胰蛋白酶消化后均未见荧光；③以pNPG为底物，βglC-Ag43表面展示酶蛋白的酶活为0.37 U/mL。

【结论】本研究成功构建了基于自转运蛋白Ag43的细菌表面展示载体，HPV16L1蛋白通过构建的四种表面展示载体均可表达并展示在大肠杆菌表面，且仅保留α-螺旋和β-桶状结构域的Ag43/700的表面展示效果最优。利用Ag43/700表面展示载体，对EGFP、NB及βglC三种外源蛋白进行细菌表面展示及功能表征，验证了该载体的通用性。

【Objective】Autotransporter is one of the key pathways that Gram-negative bacteria extensively used to secrete virulence and adhesion factors to the bacterial cell surface or into their surroundings. The AT secretion process combines the passage across the inner and the outer membrane in two consecutive steps and is used as a biotechnological tool to display recombinant proteins on the bacterial cell surface. Among all the autotransporters, the surface display system based on Ag43 is believed to be an ideal system to display foreign proteins. However, it remains elusive whether retaining the passenger domain or how many amino acids from the Ag43 passenger domain that need to be retained to achieve the optimal surface display. So the purpose of this thesis is to construct an Ag43 surface display system containing various lengths of passenger domain for an optimal bacterial cell surface display of foreign protein HPV16L1. Furthermore, three different foreign proteins: green fluorescent protein (EGFP), nanobody (NB) and β- glucosidase（βglC) were used to detect the functional expression and to examine the universality of the Ag43 surface display vector.

【Methods】(1) Construction of four Ag43 surface display vectors (Ag43/138、Ag43/551、Ag43/552、Ag43/700) by PCR and subclone. (2) Construction of foreigners-Ag43 fusions by PCR and subclone. (3) Foreigners -Ag43 fusion proteins expression and SDS-PAGE analysis. (4) Trypsin digestion of surface-exposed foreigners. (5) Detection of the function of EGFP, NB and βglC.

【Results】(1) Sequencing results showed that Ag43 surface display vectors and HPV16L1-Ag43 fusions were constructed successfully. SDS-PAGE analysis showed that HPV16L1-Ag43 fusion proteins can be expressed after IPTG induction and the protein content is reduced when the cells were treated with trypsin, especially for HPV16L1-Ag43/700 whose content is reduced drastically. (2) Sequencing results showed that EGFP-Ag43/700, NB-Ag43/700 and βglC-Ag43/700 fusions were constructed successfully too. SDS-PAGE analysis showed that three fusion proteins can be expressed after IPTG induction and the protein amount is reduced when the cells were tread with trypsin. After examining the protein function: 1) The fluorescence of EGFP-Ag43 fusion can be observed, while there was no fluorescence signal without IPTG induction. 2)NB displayed on the bacterial surface can recognize EGFP and produce fluorescence signals under laser. There is no fluorescence signal without IPTG induction or after trypsin digestion. 3)Using pNPG as a substrate, the enzyme activity of βglC on the cell surface is about 0.37 U/mL.

【Conclusion】Various Ag43 surface display systems were successfully constructed and can be used to successfully display the HPV16L1. This work showed that Ag43/700 comprising only the α-helix and the β-barrel is optimal for bacterial surface display. Moreover, three different foreign proteins EGFP, NB and βglC can be functionally displayed on the bacterial cell surface using Ag43/700, which verified the universality of the Ag43 surface display system.