Oral immunization of mice with vaccine of attenuated Salmonella typhimurium expressing Helicobacter pylori urease B subunit

Objective To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacterpylori （H. pylori） B subunit （UreB） and to determine whether it could be used as an oral vaccine against H. pylori infection. Methods Using genomic DNA of H. pylori Sydney strain （SS1） as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LBS000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups （including body weight, gastric inflammation, etc.） were also collected. Results The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H, pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-T and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed. Conclusion The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.     

Oral immunization of mice with attenuated Salmonella typhimurium expressing Helicobacter pylori urease B subunit

Objective To establish attenuated Salmonella typhimurium producing Helicobacter pylori (H. pylori) urease subunit B （UreB） and determine whether it could be used as an oral vaccine against H. pylori. Methods H. pylori (SS1 strain) UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector pTC01 after sequencing, and then transformed into attenuated Salmonella typhimurium SL3261 to acquire SL3261/pTC01- UreB. The expression of H. pylori UreB in SL3261 was detected by Western blot. Twelve weeks after oral immunization of mice,antibody responses were evaluated using serum and intestinal fluid by ELISA assay. Interferon- γ （IFN- γ） and interleukin- 10 （IL- 10） in the supernatant of spleen cells culture were also assessed by ELISA. In vitro stability of pTC01- UreB plasmid in SL3261 was confirmed by growing in Luria Broth (LB) medium to 80 generations.Results The UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB as evidenced by sequence analysis. Enzyme digestion revealed that the correct pTC01- UreB was obtained. Western blot showed that a 61kDa protein was expressed in SL3261/pTC01- UreB, which could be recognized by anti- H. pylori UreB antiserum. After 80 generations of continuous culture, the recombinant plasmid pTC01- UreB was stable in SL3261 and had no obvious toxicity. Multiple oral immunizations with SL3261/pTC01- UreB could significantly induce H. pylori- specific mucosal IgA response as well as serum IgG response. Moreover, there were significant increases of IFN- γand IL- 10 in the SL3261/pTC01- UreB group. Finally, no obvious side effects for mice and no change in gastric inflammation were observed.Conclusion Attenuated Salmonella typhimurium expressing H. pylori UreB may be used as oral vaccine against H. pylori infection.     

表达幽门螺杆菌过氧化氢酶的减毒沙门氏菌疫苗株的构建及其免疫保护作用的观察

目的 探讨表达幽门螺杆菌（Hp)过氧化氢酶（KatA)的减毒鼠伤寒沙门氏菌疫苗株在防御Hp感染中的作用。方法 构建表达KatA的重组质粒,用IPTG进行诱导表达,再将其转入减毒鼠伤寒沙门氏菌SL3261株中构建成口服活疫苗株,经口服免疫C57BL/6小鼠,并以Hp翻尼株进行攻击,用快速尿素酶试验和Hp定量培养对胃粘膜中的Hp进行检测。结果 SDS-PAGE凝胶电泳上显示一条相对分子质量约79000的新生蛋白带,占细菌总蛋白的19%,并能与抗谷胱甘肽-s-转移酶（GST）抗体发生特异性反应,动物实验结果显示;免疫小鼠能有效防御Hp的感染,结论 表达KatA的减毒沙门氏菌疫苗株能诱导抗Hp保护性免疫反应,有望在Hp感染及其相关性疾病的防治中发挥积极作用。     

幽门螺杆菌尿素酶减毒鼠伤寒杆菌疫苗诱导小鼠粘膜免疫应答的研究

目的:观察口服减毒鼠伤寒杆菌活菌重组疫苗后小鼠的粘膜免疫应答状况。方法:将已构建成功的表达幽门螺杆菌(H.pylori)尿素酶B亚单位(UreB)的重组减毒鼠伤寒杆菌SL3261/pTC01-UreB口服免疫Balb/c小鼠,12周后检测肠液和血清中的特异性抗体反应。结果:疫苗组小鼠的肠液和血清中可分别检测到针对UreB的特异性抗体IgA和IgG,病理学检查显示疫苗组小鼠较对照组小鼠胃粘膜炎症程度差异无统计学意义。结论:表达H.pyloriUreB的减毒鼠伤寒杆菌SL3261/pTC01-UreB能够诱导小鼠产生抗H.pylori的粘膜免疫,可用作抗H.pylori感染的口服疫苗。     

幽门螺杆菌中性粒细胞激活蛋白DNA疫苗的构建及其免疫保护作用

目的:构建携带幽门螺杆菌中性粒细胞激活蛋白(Hp neutrophil-activating protein,Hp-NAP)基因(napA)活减毒鼠伤寒沙门菌重组DNA疫苗,初步观察其对慢性Hp感染的免疫保护作用.方法:应用基因工程技术扩增全长napA,测序并经同源性分析后,将其亚克隆入真核表达载体pIRES,鉴定正确后将重组质粒转化活减毒鼠伤寒沙门菌构建Hp-NAP口服DNA疫苗.口服Hp-SS1建立SS1长期感染小鼠模型,30周后随机均分为3组,每组各5只.治疗组予109cfu/0.4 ml疫苗菌灌胃,1次/周×3周;2个对照组分别予等体积生理盐水或空白质粒.末次免疫4周后行快速尿素酶检测,ELISA测定血清抗体效价.结果:重组真核表达质粒pIRES-napA成功转化活减毒鼠伤寒沙门菌SL7207;所克隆napA与GenBank中SS1-na-pA核苷酸和蛋白质的同源性均＞98%.免疫后4周治疗组75%(3/4)小鼠快速尿素酶检测阴性,对照组均阳性,差异显著(P＜0.05);治疗组血清抗Hp-NAP抗体效价明显升高.结论:成功构建了具有较好免疫保护作用的Hp-NAP口服重组DNA疫苗,为进一步研制多价抗Hp核酸疫苗奠定了基础.     

携带ePNP基因的减毒伤寒沙门菌的构建及目的基因在胰腺癌细胞中的表达

目的构建含有pEGFP-C1-PNP质粒的减毒鼠伤寒沙门菌SL3261菌株,检测其在胰腺癌细胞BxPc-3中的表达。方法 PCR扩增得到PNP基因,构建真核表达载体pEGFP-C1-PNP,并进行酶切、PCR和测序鉴定。利用电转化法将重组质粒转入减毒鼠伤寒沙门菌SL3261,进行形态学和表面抗原稳定性检测。含质粒SL3261菌株与BxPc-3细胞体外共培养,利用荧光显微镜观察和RT-PCR检测绿色荧光蛋白的表达和PNP基因的转录。结果目的基因正确连接pEGFP-C1中,并成功转入减毒鼠伤寒沙门菌,感染细胞亦检测到目的基因的表达。结论成功构建了能携带ePNP基因真核表达质粒的SL3261菌株,且该基因能在胰腺癌细胞中正确表达。     

Immunogenicity of recombinant attenuated Salmonella typhimurium expressing a 45-peptide hybrid antigen gene of Plasmodium falciparum

Malariaisoneofthemajortropica1diseasesaffectinghumantoday,contributinggreatlytothemortalityrateinthedevelopingworld-Sincenoef-fectivetreatmentcouldsavethelifeofsomanypeople,thereisanurgentnecessitytodevelopaneffectivemalariavaccine.Nowresearcheshavebeentoncentratedonthestage-specificantigensofsporozoites,merozoites,exoerythrocyticandery-throcyticstagesandgametocytes.Monovalentvac-cineshavebeenprovedtohavelowprotectiveim-munity['],sopolyvalentvaccines,whichincludeseveralepitopesofdifferentantig…     

Anti-angiogenesis Effect on Glioma of Attenuated Salmonella Typhimurium Vaccine Strain with flk-1 Gene

To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3, 1-flk1 was constructed and electro-transfected into live attenuated Salmonella typhimurium strain SL7207. Mouse models of intracranial G1261 glioblastoma were treated with an orally administered attenuated Salmonella typhimurium expressing flk-1 gene. The survival period was recorded and vessel density was observed by immunofluorescence. CTLs activity was measured by MTT assay. Our results showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could significantly inhibit glioblastoma growth, reduce vessel density, prolong the survival period and improve the survival rate in these mice. The flk-1 specific CTLs activity was increased obviously after the vaccination. Our study showed that attenuated Salmonella typhlmurium vaccine strain expressing flk-1 gene could break peripheral immune tolerance a in glioma gainst this self-antigen and kill endothelial cells by the orally administered vaccine and can be used for both prophylactic and therapeutic purposes.     

幽门螺杆菌尿素酶B亚单位基因在减毒鼠伤寒杆菌中的表达

目的：制备能表达幽门螺杆菌（Ｈｐ）尿素酶Ｂ亚单位（ＵｒｅＢ）的减毒鼠伤寒杆菌,初步确定是其否可用作抗ＨＰ的口服疫苗。方法应用ＰＣＲ扩增ＨＰＵｒｅＢ基因片段,测序后克隆入原核表达载体ＰＴｃ０１,转化ＬＢ５０００修饰后再转化ＳＬ３２６１,得到重组的减毒鼠伤寒杆菌ＳＬ３２６１／ＰＴｃ０１－ＵｒｅＢ。应用抗ＨＰ菌体蛋白兔血清行Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测ＵｒｅＢ在ＳＬ３２６１中的表达,ＳＬ３２６１／ＰＴｃ     

携带flk-l的减毒沙门疫苗菌抗胶质瘤血管生成的研究

目的观察表达鼠血管内皮生长因子受体2(VEGFR2,flk-1)的重组减毒鼠伤寒沙门疫苗菌对胶质瘤荷瘤小鼠的抗肿瘤血管及肿瘤生长抑制作用。方法构建真核表达载体pcDNA3 1.-flk-l,通过电转化法将pcDNA3 1.-flk-1导入减毒鼠伤寒沙门菌SL7207中,经由胃管饲于C57BL/6J小鼠,对胶质瘤荷瘤小鼠进行免疫预防及治疗。通过观察免疫动物的生存期,免疫荧光法检测肿瘤血管密度,评价重组疫苗菌的抗血管及肿瘤生长抑制作用。分离免疫小鼠的脾细胞,分析重组疫苗菌免疫后小鼠体内的特异性细胞毒性T细胞(CTL)应答。结果重组疫苗菌免疫能够明显减少肿瘤血管密度,延缓胶质瘤的生长,延长小鼠生存期,获得明显的抗肿瘤效果。重组疫苗菌免疫后可诱导小鼠脾淋巴细胞产生针对flk-l的CTL活性。结论表达鼠flk-l的重组减毒鼠伤寒沙门疫苗菌经口服免疫,可打破小鼠对于自身抗原flk-1的免疫耐受,诱导小鼠产生抗flk-1的特异性免疫反应,特异性杀伤肿瘤血管内皮细胞,预防和治疗小鼠胶质瘤。     

Immune responses in mice to oral administration of attenuated Salmonella typhimurium expressing Helicobacter pylori urease B subunit by a lavage technique

Objective： To determine whether attenuated Salmonella typhimurium producing Helicobacter pylori （Hp） urease subunit B（UreB）can elicit specific immune responses against Hp in mice tested by a lavage technique. Methods： Attenuated Salmonella typhimurium producing Hp UreB immunized orally Balb/c mice twice at a 3-week interval. After 12 weeks, mice intestinal secretions were obtained without harm by administering a lavage solution intragastrically. The mice intestinal secretions of immune group were also directly washed out after the mice were killed. The antibody responses were evaluated by using serum and intestinal fluid with ELISA assay. Results： The multiple oral immunizations with SL3261/pTC01-UreB induced significantly Hp-specific mucosal IgA response as well as serum IgG response. The IgA was also consistently higher in the intestinal fluid obtained by the lavage solution than by direct washout. In addition, no obvious side effects and changes in gastric inflammation were observed in mice. Conclusion： The attenuated Salmonella typhimurium expressing Hp UreB may be used as an oral vaccine against Hp infection. And the lavage technique is an ideal method in the study of mucosal immune responses.     

表达人幽门螺杆菌外膜蛋白的穿梭质粒的构建及在耻垢分枝杆菌中的表达

目的建立表达人幽门螺杆菌外膜蛋白重组耻垢分枝杆菌疫苗株，为进一步应用重组耻垢分枝杆菌疫苗防治幽门螺杆菌感染打下基础。方法利用聚合酶链反应（Polymerase chain reaction，PCR）扩增的幽门螺杆菌的外膜蛋白528编码基因全长片段，克隆入pET32a（＋），鉴定无误后，再亚克隆入大肠杆菌一分枝杆菌穿梭质粒pLA71，构建pLA71-omp载体，将pLA71-omp电转化耻垢分枝杆菌，经卡那霉素筛选后，抽提质粒用PCR法鉴定，Westernblot检测omp的表达及活性。结果从幽门螺杆菌基因组中扩增出约528bp的基因片段。所构建重组体阳性克隆经酶切和PCR鉴定与预期结果一致，测序结果确证了插入片段的正确性，Westernblot检测说明幽门螺杆菌的外膜蛋白528编码基因在分枝杆菌中获得了表达并具有良好的免疫原性。结论成功构建了幽门螺杆菌外膜蛋白528编码基因大肠杆菌一分枝杆菌穿梭表达质粒。该质粒能在E.coli／MS间进行穿梭，并在耻垢分枝杆菌中表达。     

恶性疟口服活菌苗的免疫原性及安全性的初步研究

在减毒鼠伤寒沙门氏菌ＳＬ３２６１中表达了人工合成的恶性疟杂合４５肽抗原基因，并在ＢＡＬＢ／ｃ小鼠及家兔中诱发出一定水平的特异性体液免疫及细胞免疫。在减毒菌中表达的融合抗原ＧＺ－Ａ可特异地识别兔抗ＧＺ－Ａ血清、鼠抗Ｐｆ血清及恶性疟患者血清，滴度分别为１：６４００、１：１０２４０及１：４。小鼠口服（ｐｏ）重组疫苗图ＳＬ３２６１（ｎＷＲＡ）后，细菌在肠道至少可寄生１月以上，且在体外仍然可以表达目的抗原。静脉注射（ｉｖ）活菌后首日可见菌血症。１０￣８ｃｆｕ量全身免疫（ｉｐ、ｉｖ）后可致小鼠死亡。ｉｐ、ｉｖ组脾脏肿大１～２倍，肝脏轻度肿大，而ｐｏ组则未见明显改变。清理切片显示肝脏轻度充血，汇管区有淋巴细胞聚集，枯否氏细胞轻度增生等；脾小体增生扩大，巨细胞明显增生等，上述改变以ｉｐ及ｉｖ组较为明显。结果说明，ＳＬ３２６１（ｐＷＲＡ）所表达的恶性疟原虫杂合抗原可特异地被抗Ｐｆ抗体所识别，活菌苗口服后可在肠道较长久地寄生，且不引起明显的毒副作用。     

幽门螺杆菌尿素酶基因ureB植物双元表达载体的构建

目的:构建含有人幽门螺杆菌(H.pylori,Hp)ureB基因的植物表达载体.方法:采用高保真PCR技术从质粒pMED-ureB中扩增出ureB基因,构建质粒pUC18-ureB,再将pUC18-ureB酶切,将ureB基因与质粒pBI121连接.结果:构建的PBI121-ureB经PCR、酶切鉴定和测序分析证明,插...     

pIRES2-EGFP-4-1BBL质粒的导入对减毒沙门菌生物学行为的影响

目的探讨真核表达质粒pIRES2-EGFP-4-1BBL转入对减毒鼠伤寒沙门菌SL3261生物学行为的影响。方法将真核表达质粒pIRES2-EGFP-4-1BBL通过两步电转化获得含pIRES2-EGFP质粒的SL3261,接种于LB培养基中观察其生长特性、革兰染色观察其形态和血清凝集试验检测其表面抗原变化。利用该重组菌体外感染HepG2细胞,观察其对细胞的侵袭能力的影响。结果含pIRES2-EGFP-4-1BBL质粒SL3261在体外生长繁殖较原细菌慢,革兰染色呈阴性丝状菌体,含pIRES2-EGFP-4-1BBL质粒的SL3261 A-F多价、O4菌体和H1鞭毛抗原和SL3261完全一致;体外感染HepG2能力,SL3261为201±46 CFU/200HepG2细胞,SL3261-pIRES2-EGFP为163±37,而SL3261-pIRES2-EGFP-4-1BBL为158±32,3组间的差异无统计意义（P〉0.05）。结论导入pIRES2-EGFP-4-1BBL质粒对SL3261的生长和形态有部分影响,而表面抗原以及侵入HepG2细胞的功能不变,该结果为含pIRES2-EGFP-4-1BBL质粒SL3261疫苗菌的进一步研究奠定了实验依据。     

荷瘤小鼠口服重组减毒鼠伤寒沙门菌后瘤体内富集效果的观察

目的观察重组减毒鼠伤寒沙门菌作为基因载体在人涎腺腺样囊性癌裸鼠移植瘤中的富集情况和对外源基因的呈递能力。方法以绿色荧光蛋白基因(GFP)为报告基因,以减毒鼠伤寒沙门菌SL7207为转基因载体,分别构建原核启动GFP表达的重组减毒鼠伤寒沙门菌SL7207-pUC-GFP和真核启动GFP表达的重组减毒鼠伤寒沙门菌SL7207-pEG-FP-N1。原核菌SL7207-pUC-GFP在体外连续传代,观察GFP基因表达的稳定性;同时,对荷人涎腺腺样囊性癌皮下移植瘤裸鼠模型口服给予原核菌SL7207-pUC-GFP(0.1mL,1×109cfu/mL),在口服后24h、48h、5d、10d、20d、30d处死荷瘤裸鼠,获取肝、脾及肿瘤组织并制成匀浆液进行重组菌培养及GFP表达检测,观察重组菌在瘤体细胞内富集情况。对荷瘤裸鼠模型口服给予真核菌SL7207-pEGFP-N1,5d后取肝、脾及肿瘤组织进行冰冻组织切片,荧光显微镜下观察GFP的表达,了解重组菌携带外源基因在肿瘤细胞内的表达。结果携带GFP原核表达的重组减毒鼠伤寒沙门菌SL7207-pUC-GFP在体外连续传代10次未见表达减少或缺失。荷瘤裸鼠口服原核表达GFP基因重组菌SL7207-pUC-GFP菌液实验表明,重组菌SL7207-pUC-GFP在肝、脾及肿瘤组织中能长期存活,以肿瘤组织中聚集明显(P<0.05)且维持时间较长。荷瘤裸鼠口服真核表达GFP基因重组菌SL7207-pEGFP-N1菌液实验表明,相对肝脏和脾脏组织,外源基因在肿瘤细胞内表达量最高。结论重组减毒鼠伤寒沙门菌可以在瘤体细胞内富集存活,并且携带的外源基因可以释放到肿瘤细胞内表达,具有作为基因治疗载体的双重优势。     

表达幽门螺杆菌过氧化氢酶的无抗性减毒鼠伤寒沙门氏菌株的构建

OBJECTIVE: To construct a non-resistant attenuated Salmonella typhimurium (S.typhimurium) strain capable of expressing Helicobacter pylori (Hp) catalase. METHODS: After PCR amplification, the gene fragment encoding Hp catalase was inserted into the expression vector pYA248 containing asd gene, and the recombinant vector was then introduced into the host S.typhimurium strain X4072 depleted of genes encoding adenylate cyclase (delta cya), cyclic adenosine monophosphate receptor protein (delta crp) and aspartate-beta-semialdehyde dehydrogenase (delta asd). Bridged enzyme-linked immunosorbent assay (ELISA) was employed to measure the antigenicity of the catalase expressed in the sonicate and culture supernatant. According to Meacock's method and with the assistance of the growth curve, the stability of the recombinant strain was evaluated. A half lethal oral dose test was conducted to evaluate the safety of recombinant strain. RESULTS: S.typhimurium X4072 (pYA248-CAT) with expected capacity was successfully constructed, and bridged ELISA demonstrated higher catalase levels in the culture supernatant than in the sonicate of the recombinant strain X4072 (pYA248-CAT). After the strain was passaged for 100 generations without selection pressure, all the randomly selected colony of the recombinant strain grew well with positive catalase antigenicity as identified by ELISA. The growth curve of the recombinant strain showed comparable growth status of the 2 strains X4072 (pYA248) and X4072 (pYA248-CAT). The survival rate of C57BL/6 mice was 100% 30 d after oral administration of 1.0x10(10) cfu X4072 (pYA248-CAT). CONCLUSION: Non-resistant S. typhimurium vaccine X4072 (pYA248- CAT) is constructed successfully, which is stable in vitro and safe as confirmed by animal experiment. This vaccine provides a new candidate for viable oral vaccine against Hp infection.     

幽门螺杆菌儿童分离株中性粒细胞激活蛋白克隆及表达序列

目的：从幽门螺杆菌临床儿童分离株GZCH1基因组扩增中性粒细胞激活蛋白（NAP）基因,克隆入T载体,并亚克隆入表达载体pGEX-4T-1,进行测序及基因比对分析,为幽门螺杆菌疫苗研制奠定基础.方法：根据GenBank中幽门螺杆菌NAP序列,设计一对特异性引物扩增幽门螺杆菌临床儿童分离株NAP全长基因,与T载体连接,转化大肠杆菌DH5α,提取质粒进行酶切、测序鉴定,经EcoR Ⅰ、Not Ⅰ双酶切后与做相应酶切的pGEX-4T-1连接,转化大肠杆菌BL21,提取质粒进行双酶切鉴定,IPTG诱导重组蛋白表达,并对基因进行测序及比对分析.结果：以幽门螺杆菌儿童分离株GZCH1为模板,成功扩增了NAP基因,基因大小为435 bp,重组pGEX-4T-1-NAP双酶切鉴定可见目的片段,测序结果显示NAP在正确读框中,序列比对分析显示其与幽门螺杆菌J99株氨基酸一致性达99.2％,IPTG诱导后,pGEX4T-1-NAP/BL21在相应分子量（42.8 kD）可见融合蛋白的表达.幽门螺杆菌儿童分离株GZCH1 NAP序列已登录GenBank（登录号：GU301881）.结论：从幽门螺杆菌临床儿童分离株GZCH1中成功克隆了NAP基因,并获得重组蛋白的表达,为NAP幽门螺杆菌疫苗研制奠定了良好的基础.     

两种表达鼠精子受精素β蛋白的减毒沙门氏菌疫苗株的稳定性分析

目的 了解表达鼠精子抗原受精素β亚单位(Fertilin βsubumit,fβ)的两种重组减毒沙门氏菌疫苗株X4632(pFEC)和X4550(pFEC)的生长特性和体内外携带重组质粒的稳定性。方法 将两种重组沙门氏菌疫苗株X4632(pFEC)和X4550(pFEC)体外传代培养，观察比较其生长特性和所含重组质粒的稳定性；将该两种重组菌口服免疫BAIB／c雄性小鼠，观察重组菌侵入小鼠Peyer结、肠系膜淋巴结和脾脏等组织内繁殖生长情况和所回收重组菌携带质粒的阳性率。结果 该两种无抗药性的重组菌株在体内、外营养选择压力下均可较稳定携带重组质粒传代繁殖，X4550(pCFL)在体内可较稳定地定植于Peyer结、肠系膜淋巴结和脾脏；X4632(pCFL)可较稳定地定植于Peyer结，均可稳定表达被Fβ单抗识别的重组Fβ蛋日。结论 X4632(pFEC)、X4550(pFEC)疫苗株可作为递呈精子抗原的活菌载体。     

mFSHR基因N端片段的序列分析、原核表达载体构建及其表达

目的：克隆小鼠卵泡刺激素受体（FSHR）基因N-端部分片段（28—90aa）（mFSHRn）;预测其编码蛋白作为候选避孕疫苗的可行性;构建原核表达重组质粒,并在大肠杆菌中表达。方法：提取小鼠睾丸组织总RNA,利用逆转录．聚合酶链反应（RT—PCR）技术反转录成cDNA,按照GeneBank中小鼠FSHRN．端序列设计引物,扩增基因片段并插入pET32a载体,测序鉴定后做生物信息学分析;质粒转化E．coli BL21（DE3）感受态菌株诱导表达蛋白。结果：扩增片段长度为186bp,测序结果与预期结果完全一致,生物信息学分析其编码蛋白具有良好的抗原性;重组原核表达质粒经鉴定获得正确重组子并诱导表达。结论：mFSHRn蛋白可作为良好的候选避孕疫苗,成功构建了pET32a—mFHSRn原核表达重组质粒,获得可表达mFHSRn的大肠杆菌菌株,为后续的相关研究奠定了基础。