表达H5N1血凝素HAl重组乳酸菌的构建及其免疫效力的研究
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摘要
为研制安全、有效、方便操作的口服免疫禽流感活载体疫苗,本试验从健康成年鸡回盲肠黏膜处分离筛选到一株具有较强耐酸能力及较好上皮细胞粘附能力的鸡源乳杆菌DLD17.通过分子生物学的方法,我们构建了重组H5N1血凝素乳酸乳球菌表达质粒pMG36EH和重组H5N1血凝素乳酸乳杆菌表达质粒pLEM415-pH,通过电击转化分离的鸡源乳酸菌DLD17,乳酸乳球菌1363和嗜酸乳杆菌4356。我们获得了稳定表达禽流感血凝素蛋白的三株重组乳酸菌,即重组乳酸乳球菌LL36EH、重组鸡源乳杆菌DLD17-pH和重组嗜酸乳杆菌4356-pH.首先将三株重组乳酸菌口服免疫小鼠,及其产生的免疫效果进行了评价和比较。结果发现,重组鸡源乳杆菌DLD17-pH在诱导机体产生体液免疫、细胞免疫的效果最好。因此我们选用重组鸡源乳杆菌DLD17-pH口服免疫雏鸡并对其免疫效果进行了评价。结果证实,重组鸡源乳杆菌DLD17-pH能够在雏鸡的盲肠内定植并稳定繁殖。它不但较早、较快的诱导呼吸道、消化道黏膜禽流感特异性IgA抗体的产生,而且也能在一定程度上诱导全身性禽流感特异性IgG抗体的分泌。除此之外,重组鸡源乳杆菌DLD17-pH还能激活机体细胞免疫和先天性免疫反应,增强机体免疫防御能力。因此,重组鸡源乳杆菌DLD17-pH口服疫苗的研制,为预防和控制禽流感的传播提供了新的研究思路,为宿主乳酸菌作为活载体递送表达系统理论研究和实践应用奠定了基础。具体分为以下四个方面：
1.鸡源乳酸菌的分离、鉴定与筛选
首先从健康成年鸡盲肠黏膜处分离了22株革兰氏阳性菌株,设计细菌16s-rRNA细菌特异性引物,扩增每株细菌的16s片段,并与菌株信息库比对确定菌株的种属。再次筛选从而确定了5株鸡源乳酸菌(DLD-1、DLD-5、DLD-9、DLD-13、DLD-17)用作受体转化菌株备用。同时结合菌株生化发酵试验,鉴定了五株鸡源乳酸菌的属种。此外,我们还对五株鸡源乳杆菌的耐酸性、对Caco-2细胞粘附力以及抗生素敏感性进行测定。通过乳酸乳球菌1363和嗜酸乳杆菌4356作为电击转化阳性对照受体菌株,以乳酸乳球菌质粒pMG36E和乳酸杆菌质粒pLEM415为空载体质粒,进行感受态的制备和电转条件的摸索,确定了乳酸菌株转化的最优电击转化方案。获得最优转化方案后,对五株鸡源乳杆菌做电击转化筛选试验,筛选能够接受外源质粒的鸡源乳杆菌。试验结果证明,我们分离并筛选到的五株菌株中,一株为粪肠乳球菌DLD-1,一株为发酵乳酸杆菌(DLD-5),三株为唾液乳酸杆菌(分别为DLD-9、DLD-13、DLD-17)。通过电击转化筛选试验,我们得到一株鸡源乳杆菌适合接受外源性质粒,即唾液乳酸杆菌DLD17。
2.表达H5N1血凝素重组乳酸菌的构建及其验证
我们首先设计含有限制性内切酶SalI和HindⅢ的禽流感血凝素特异性上下游引物HAF1和HAF2,PCR扩增H5N1亚型高致病性禽流感(AIV)血凝素部分片段HAI,连接pMD19T载体,测序验证后,用限制性内切酶SalI和HindⅢ分步酶切表达质粒pMG36E和克隆质粒pMD19T-H,回收并连接载体骨架pMG36E与目的片段HA1,连接获得了乳酸乳球菌表达质粒pMG36E-H;通过电击转化的方法,获得重组乳酸乳球菌1363-EH(即含pMG36E-H),经SDS-PAGE和Western blot检验；结果表明,43KDa的血凝素蛋白在乳酸乳球菌内正确表达。同以上方法,我们也构建了重组血凝素乳酸杆菌的表达质粒pLEM415-pH。我们设计启动子引物P1和P2,扩增乳酸菌的乳酸脱氢酶(LDH)启动子P；血凝素引物HAF1和HAF2扩增乳杆菌血凝素片段HA1,通过分子生物学酶切连接的方案,将启动子P和血凝素基因HA1依次连接到乳杆菌空载体表达质粒pLEM415上,从而获得了重组乳酸杆菌表达质粒pLEM415-pH。然后通过电击转化的方法,转入到鸡源乳杆菌DLD17和嗜酸乳杆菌4356菌株内,获得两株重组血凝素基因的乳酸杆菌,分别是重组鸡源乳杆菌DLD17-pH和重组嗜酸乳杆菌4356-pH。通过蛋白电泳和Western-blot验证,结果表明,血凝素HA1可以在两株重组乳酸杆菌中表达。总之,我们获得了三株表达禽流感血凝素的重组乳酸菌,分别为重组乳酸乳球菌LL36EH、重组鸡源乳杆菌DLD17-pH和重组嗜酸乳杆菌4356-pH,为下面表达禽流感血凝素重组乳酸菌口服疫苗的研究奠定基础。
3.三株重组乳酸菌口服免疫小鼠及其免疫效果的研究
运用第二章转化获得的三株重组乳酸菌(1363-EH、DLD7-pH、LA4356-pH)口服免疫小鼠,研究小鼠呼吸道、消化道局部和全身免疫抗体水平的变化；以及分离免疫小鼠脾脏淋巴细胞,分析CD4+/CD8+、CD19+细胞亚群的变化；并在体外用血凝素HA1抗原特异性刺激来研究脾淋巴细胞增殖试验和细胞因子IFN-γ和IL-4的分泌情况。结果发现,1363-EH、4356-pH、D1D7-pH三株重组乳酸菌口服免疫小鼠,免疫后1周,其均可显著诱导肠道禽流感特异性IgA抗体的分泌；随着时间的加长,LL36EH、4356-pH两株免疫小鼠组的特异IgA抗体水平下降,而DLD-pH株免疫产生的禽流感抗体表达随时间增加处于稳定水平。由此可见,三株乳酸菌均能诱导局部黏膜免疫应答。而在气管内,仅有鸡源乳杆菌DLD17-pH免疫组诱导了特异性IgA的显著性分泌,其出现的时间比肠道晚一周。在全身性免疫反应的检测中,我们发现三株重组乳酸菌免疫后小鼠,血清内特异性IgG抗体和HI效价都较低,其中DLD17-pH免疫组小鼠抗体滴度最高为4.5(Log2).小鼠脾脏淋巴细胞激增殖试验和CD4+/CD8+、CD19+细胞亚群细胞的变化表明重组乳酸菌可在体内激活T淋巴细胞和B淋巴细胞；其中致敏的T淋巴细胞,主要以Th2型辅助性T细胞的增殖有关,而三株之间没有显著差异。由以上结果可知,三株表禽流感血凝素的重组乳酸菌,口服免疫小鼠后,在一定程度上均可刺激机体发生禽流感特异性体液、细胞免疫反应；但因菌株来源、特性的不同,免疫效果差别较大。重组鸡源乳杆菌DLD17-pH在诱导机体产生体液免疫、细胞免疫的效果最好,尤其可以有效诱导肠道、呼吸道黏膜特异性IgA抗体的分泌,更适合应用于家禽禽流感的黏膜免疫。
4.重组鸡源乳杆菌DLD17-pH口服免疫雏鸡效果的研究
运用第三章筛选的优良重组鸡源乳杆菌DLD17-pH口服免疫雏鸡,研究雏鸡呼吸道、消化道局部和全身免疫抗体水平的变化,并对重组鸡源乳杆菌DLD17-pH在雏鸡肠道内的定植情况进行检测；同时与禽流感灭活疫苗的免疫效果进行比较。结果发现：口服重组鸡源乳杆菌DLD17-pH,不但能诱导空肠、盲肠黏膜局部特异性IgA的产生,还能够诱导气管处黏膜特异性IgA的产生。此外,口服重组鸡源乳杆菌也能够诱导全身特异性IgG抗体的产生,但是水平较低。与皮下免疫禽流感灭活疫苗组相比,重组鸡源乳杆菌的口服免疫24h后,能显著上调消化道、呼吸道局部组织处的细胞因子IFN-γ、模式识别受体TLR-2、防御素AvBD-9的基因表达；而下调细胞因子IL-4基因的表达。盲肠内重组鸡源乳杆菌DLD17-pH数量的检测表明,口服免疫后,重组鸡源乳杆菌DLD17-pH能够粘附定植于盲肠黏膜处,并且在21天菌落较为稳定。总之,口服重组鸡源乳杆菌DLD17-pH不仅可以激活消化道局部组织早期细胞因子的分泌,而且在呼吸道、消化道局部以及全身均能有效的诱导禽流感特异性抗体的产生。因此口服表达重组鸡源乳杆菌DLD17-pH为家禽禽流感的预防和免疫提供了一条新的思路。
To develop a safe, effective, and convenient vaccine for the prevention of highly pathogenic avian influenza (HPAI), we separated a Lactobacillus DLD17with a strong acid capacity and better epithelial cell adhesion ability from a healthy adult chicken cecal mucosa. By using the methods of molecular biology, we have successfully constructed two recombinant expression plasmids pMG36EH (the expressing plasmid of Lactococcus lactis) and pLEM415-pH (the expressing plasmid of Lactobacillus). After transform recombinant plasmids into lactic acid bacteria Lactococcus lactis1363and Lactobacillus acidophilus4356by electroporation, we have constructed three recombinant bacterials (LL36EH, DLD17-pH and4356-pH) with stably expressing the foreign protein hemagglutininl (HA1) of HPAI virus. Immunizations were administered orally to the BALB/c mice three times at2-week intervals to evaluate the oral immune effect. Results showed that the humoral and cellular immune responses triggered by recombinant lactobacillus (DLD17-pH) were significant higher than that in other two groups. So we selected the recombinant lactobacillus (DLD17-pH) to proceed the next orally immunizations on chicken to evaluate the mucosal and systemic immune effective. The results showed that the recombinant chicken Lactobacillus (DLD17-pH) could stably colonizationand reproduction in the cecum of chickens. It was also demonstrated that the recombinant Lactobacillus (DLD17-pH) not only could triggered the production of the specific anti-HAl IgA antibody level in the mucosa of respiratory and intestinal, but also could increase the anti-HAl IgG secreation in system level. Our research also proved that DLD17-pH could activate the cellular immunity and innate immune response to enhance the immune defense capability. Therefore, DLD17-pH could be a promising oral vaccine candidate against HPAI. The development of recombinant Lactobacillus (DLD17-pH) oral vaccine not only provides a new research idea for the prevention and control of the spread of avian flu, but also laid the foundation for the application of host lactobacillus as live vector delivery system. I will discuss it from the following the four aspects.
1. Isolation, identification and screening of lactic acid bacteria in chicken
Firstly,22Gram positive strain, harvested from the cecal mucosa of health adult chicken, were identified by amplified16s-rRNA fragments of each strain of bacteria, and compared with determining strains of species and strains.Combined with the results of biochemical fermentation strains, the five species of lactic acid bacteria strains in chicken were determine as the receptor into strains. In addition, five strains of acid tolerance of Lactobacillus from chicken, Caco-2cell adhesion, and antibiotic sensitivity were determined. To determine the optimal shock transformation of lactic acid bacteria transformation program, lactococcus vector plasmid plem415blank pmg36e and Lactobacillus vector plasmid transfer lactococcus1363and4356as Lactobacillus acidophilus transformation receptor positive control strain of electric shock, competent preparation and electron conditions of trial and error. After the optimal conversion options, the five strains of Lactobacillus from chicken to do transformation filter test of electric shock, the filter can accept of exogenous plasmid of Lactobacillus from chicken. These results suggestion that isolate and filter down to five strains of strain, a milk Neisseria gonorrhoeae strains of fecal gut DLD-1, a strain of Lactobacillus fermentation (DLD-5), three for salivary Lactobacillus DLD-9, DLD-13, DLD-17, respectively. Through electric shocks into screening test, we get a strain of Lactobacillus from chicken suitable for exogenous plasmid, the salivary Lactobacillus DLD-17.
2. Construction of recombinant H5N1haemagglutinin expression of lactic acid bacteria and its verification
In this chapter, we firstly designed two primer HAF1and HAF2containing restrictive enzyme SalⅠ and HindⅢ. The part of fragment HA1in H5N1Asia type high pathogenic sexual avian influenza (AIV) blood coagulation were cloned into pmd19-T vector. After measuring sequence validation,. Linearized vector pmg36e and purpose fragment HA1with restrictive enzyme SalⅠ and HindⅢ were transfer recombinant lactic acid milk Neisseria gonorrhoeae, named1363-EH, through electric shock transformation. SDS-page and Western blot results indicate that blood coagulation pigment protein in lactic acid milk Neisseria gonorrhoeae were correctly express. As the above, we also constructed the expression plasmid of recombinant hemagglutinin Lactobacillus pLEM415-pH. To clone the promoter p of lactic acid dehydrogenation enzyme (LDH), two primer P1and P2were designed. Then promoter p and blood coagulation pigment gene HA1were connected to Bacillus vector pLEM415, constructing of recombinant lactic acid Bacillus expression pLEM415-pH. Secondly, vector pLEM415-pH were transfer through the method of transformation of electric shock, obtained two recombinant hemagglutinin gene of strains of Lactobacillus, namely4356-pH and DLD17-pH. Protein electrophoresis and Western-blot were shown that the haemagglutinin of HA1were expressed in two recombinant Lactobacillus strains. In short, we have three strains of lactic acid bacteria for recombinant expression of hemagglutinin of avian influenza, namely restructuring lactococcus1363-EH, recombinant of Lactobacillus acidophilus4356-pH and DLD17-pH, which laid a solid foundation for following expression of hemagglutinin of avian influenza research of recombinant Lactobacillus oral vaccine.
3. Immunity levels of mouse after immunized orally with three recombinant lactic acid bacteria expressing H5
Observe antibody changes of mouse respiratory and gastrointestinal tract and whole body after oral immunized with three recombinant lactic acid bacteria1363-EH, DLD7-pH and LA4356-pH. Isolate spleen lymphocytes of immunized mouse and analyze the ratio ofCD4+/CD8+, CD19+lymphocyte subpopulations. Observe proliferation and secretion level of IFN-y and IL-4lymphocytes in vitro under the stimulus of specific antigen HAL Results indicated that IgA level was significantly increased in gastrointestinal tract after oral immunizing with this three bacteria; IgA level oral immunized with LL36EH and4356-pH was decreased with the lengthening of immunized time, while IgA level of mouse immunized with DLD-pH keep stability. These show that these three bacteria can induce local mucosal immune responses. In trachea, only chicken Lactobacillus DLD17-pH group show significant specific IgA secretion, a week later than the gut. We found that IgG antibody level and HI titers in the serum mice immunes with the three recombinant lactic acid bacteria are low, immunized, it is up to4.5(the Log2) of mice immune with specific DLD17-pH. Integrated with lymphocytes proliferation test and the ratio of CD4+/CD8+,CD19+lymphocyte subpopulations, we found that recombinant lactic acid bacteria can activate T lymphocytes and B lymphocytes in the body; which is mainly caused by the proliferation of Th2type the helper T cells, whereas no significant differences between the three bacteria. To some extent, three recombinant lactic acid bacteria which can express the avian influenza hem agglutinin can stimulate specifically humeral and cellular immune response, but immune effect different because of the strain source, characteristics. DLD17-pH did best in inducing humeral and cellular immune response, particularly it can effectively induce intestinal secretion of specific IgA antibodies in the respiratory mucosa. Herein, DLD17-pH is most suitable for mucosal immunity applied to the bird flu in poultry.
4. Study on Immune effect of recombinant chicken Lactobacillus DLD17-pH in chicks immunized orally
Immune chicks orally with restructured chicken Lactobacillus DLD17-pH to research local and systemic antibody level changes in respiratory and digestive tract in chicks. Detect the colonization of DLD17-pH in chick intestinal and compare immune effects with avian influenza inactivated vaccine. The results showed that DLD17-pH can not induce the production of specific IgA in the jejunum, cecal mucosa local and trachea, but also can induce specific low-level IgG antibodies. Compared with the subcutaneous immunization of avian influenza inactivated vaccine group, DLD17-pH can significantly increase gene expression of cytokines IFN-y in the digestive and respiratory tract organizations locally, TLR-2, and AvBD, While decrease gene expression of cytokines IL-4. Increasing of DLD17-pH in the cecum shows DLD17-pH can colonize in the cecal mucosa. It keeps relatively stable on21th day. In summary, oral administration of recombinant Lactobacillus DLD17-pH can not only activate early cytokine secretion in the digestive tract local tissue, respiratory, but also can induce specific antibodies of the avian flu in whole body. Therefore, it provides a new train of thought for prevents poultry bird flu and immunity by oral immunization recombinant Lactobacillus DLD17-pH.
1.鸡源乳酸菌的分离、鉴定与筛选
首先从健康成年鸡盲肠黏膜处分离了22株革兰氏阳性菌株,设计细菌16s-rRNA细菌特异性引物,扩增每株细菌的16s片段,并与菌株信息库比对确定菌株的种属。再次筛选从而确定了5株鸡源乳酸菌(DLD-1、DLD-5、DLD-9、DLD-13、DLD-17)用作受体转化菌株备用。同时结合菌株生化发酵试验,鉴定了五株鸡源乳酸菌的属种。此外,我们还对五株鸡源乳杆菌的耐酸性、对Caco-2细胞粘附力以及抗生素敏感性进行测定。通过乳酸乳球菌1363和嗜酸乳杆菌4356作为电击转化阳性对照受体菌株,以乳酸乳球菌质粒pMG36E和乳酸杆菌质粒pLEM415为空载体质粒,进行感受态的制备和电转条件的摸索,确定了乳酸菌株转化的最优电击转化方案。获得最优转化方案后,对五株鸡源乳杆菌做电击转化筛选试验,筛选能够接受外源质粒的鸡源乳杆菌。试验结果证明,我们分离并筛选到的五株菌株中,一株为粪肠乳球菌DLD-1,一株为发酵乳酸杆菌(DLD-5),三株为唾液乳酸杆菌(分别为DLD-9、DLD-13、DLD-17)。通过电击转化筛选试验,我们得到一株鸡源乳杆菌适合接受外源性质粒,即唾液乳酸杆菌DLD17。
2.表达H5N1血凝素重组乳酸菌的构建及其验证
我们首先设计含有限制性内切酶SalI和HindⅢ的禽流感血凝素特异性上下游引物HAF1和HAF2,PCR扩增H5N1亚型高致病性禽流感(AIV)血凝素部分片段HAI,连接pMD19T载体,测序验证后,用限制性内切酶SalI和HindⅢ分步酶切表达质粒pMG36E和克隆质粒pMD19T-H,回收并连接载体骨架pMG36E与目的片段HA1,连接获得了乳酸乳球菌表达质粒pMG36E-H;通过电击转化的方法,获得重组乳酸乳球菌1363-EH(即含pMG36E-H),经SDS-PAGE和Western blot检验；结果表明,43KDa的血凝素蛋白在乳酸乳球菌内正确表达。同以上方法,我们也构建了重组血凝素乳酸杆菌的表达质粒pLEM415-pH。我们设计启动子引物P1和P2,扩增乳酸菌的乳酸脱氢酶(LDH)启动子P；血凝素引物HAF1和HAF2扩增乳杆菌血凝素片段HA1,通过分子生物学酶切连接的方案,将启动子P和血凝素基因HA1依次连接到乳杆菌空载体表达质粒pLEM415上,从而获得了重组乳酸杆菌表达质粒pLEM415-pH。然后通过电击转化的方法,转入到鸡源乳杆菌DLD17和嗜酸乳杆菌4356菌株内,获得两株重组血凝素基因的乳酸杆菌,分别是重组鸡源乳杆菌DLD17-pH和重组嗜酸乳杆菌4356-pH。通过蛋白电泳和Western-blot验证,结果表明,血凝素HA1可以在两株重组乳酸杆菌中表达。总之,我们获得了三株表达禽流感血凝素的重组乳酸菌,分别为重组乳酸乳球菌LL36EH、重组鸡源乳杆菌DLD17-pH和重组嗜酸乳杆菌4356-pH,为下面表达禽流感血凝素重组乳酸菌口服疫苗的研究奠定基础。
3.三株重组乳酸菌口服免疫小鼠及其免疫效果的研究
运用第二章转化获得的三株重组乳酸菌(1363-EH、DLD7-pH、LA4356-pH)口服免疫小鼠,研究小鼠呼吸道、消化道局部和全身免疫抗体水平的变化；以及分离免疫小鼠脾脏淋巴细胞,分析CD4+/CD8+、CD19+细胞亚群的变化；并在体外用血凝素HA1抗原特异性刺激来研究脾淋巴细胞增殖试验和细胞因子IFN-γ和IL-4的分泌情况。结果发现,1363-EH、4356-pH、D1D7-pH三株重组乳酸菌口服免疫小鼠,免疫后1周,其均可显著诱导肠道禽流感特异性IgA抗体的分泌；随着时间的加长,LL36EH、4356-pH两株免疫小鼠组的特异IgA抗体水平下降,而DLD-pH株免疫产生的禽流感抗体表达随时间增加处于稳定水平。由此可见,三株乳酸菌均能诱导局部黏膜免疫应答。而在气管内,仅有鸡源乳杆菌DLD17-pH免疫组诱导了特异性IgA的显著性分泌,其出现的时间比肠道晚一周。在全身性免疫反应的检测中,我们发现三株重组乳酸菌免疫后小鼠,血清内特异性IgG抗体和HI效价都较低,其中DLD17-pH免疫组小鼠抗体滴度最高为4.5(Log2).小鼠脾脏淋巴细胞激增殖试验和CD4+/CD8+、CD19+细胞亚群细胞的变化表明重组乳酸菌可在体内激活T淋巴细胞和B淋巴细胞；其中致敏的T淋巴细胞,主要以Th2型辅助性T细胞的增殖有关,而三株之间没有显著差异。由以上结果可知,三株表禽流感血凝素的重组乳酸菌,口服免疫小鼠后,在一定程度上均可刺激机体发生禽流感特异性体液、细胞免疫反应；但因菌株来源、特性的不同,免疫效果差别较大。重组鸡源乳杆菌DLD17-pH在诱导机体产生体液免疫、细胞免疫的效果最好,尤其可以有效诱导肠道、呼吸道黏膜特异性IgA抗体的分泌,更适合应用于家禽禽流感的黏膜免疫。
4.重组鸡源乳杆菌DLD17-pH口服免疫雏鸡效果的研究
运用第三章筛选的优良重组鸡源乳杆菌DLD17-pH口服免疫雏鸡,研究雏鸡呼吸道、消化道局部和全身免疫抗体水平的变化,并对重组鸡源乳杆菌DLD17-pH在雏鸡肠道内的定植情况进行检测；同时与禽流感灭活疫苗的免疫效果进行比较。结果发现：口服重组鸡源乳杆菌DLD17-pH,不但能诱导空肠、盲肠黏膜局部特异性IgA的产生,还能够诱导气管处黏膜特异性IgA的产生。此外,口服重组鸡源乳杆菌也能够诱导全身特异性IgG抗体的产生,但是水平较低。与皮下免疫禽流感灭活疫苗组相比,重组鸡源乳杆菌的口服免疫24h后,能显著上调消化道、呼吸道局部组织处的细胞因子IFN-γ、模式识别受体TLR-2、防御素AvBD-9的基因表达；而下调细胞因子IL-4基因的表达。盲肠内重组鸡源乳杆菌DLD17-pH数量的检测表明,口服免疫后,重组鸡源乳杆菌DLD17-pH能够粘附定植于盲肠黏膜处,并且在21天菌落较为稳定。总之,口服重组鸡源乳杆菌DLD17-pH不仅可以激活消化道局部组织早期细胞因子的分泌,而且在呼吸道、消化道局部以及全身均能有效的诱导禽流感特异性抗体的产生。因此口服表达重组鸡源乳杆菌DLD17-pH为家禽禽流感的预防和免疫提供了一条新的思路。
To develop a safe, effective, and convenient vaccine for the prevention of highly pathogenic avian influenza (HPAI), we separated a Lactobacillus DLD17with a strong acid capacity and better epithelial cell adhesion ability from a healthy adult chicken cecal mucosa. By using the methods of molecular biology, we have successfully constructed two recombinant expression plasmids pMG36EH (the expressing plasmid of Lactococcus lactis) and pLEM415-pH (the expressing plasmid of Lactobacillus). After transform recombinant plasmids into lactic acid bacteria Lactococcus lactis1363and Lactobacillus acidophilus4356by electroporation, we have constructed three recombinant bacterials (LL36EH, DLD17-pH and4356-pH) with stably expressing the foreign protein hemagglutininl (HA1) of HPAI virus. Immunizations were administered orally to the BALB/c mice three times at2-week intervals to evaluate the oral immune effect. Results showed that the humoral and cellular immune responses triggered by recombinant lactobacillus (DLD17-pH) were significant higher than that in other two groups. So we selected the recombinant lactobacillus (DLD17-pH) to proceed the next orally immunizations on chicken to evaluate the mucosal and systemic immune effective. The results showed that the recombinant chicken Lactobacillus (DLD17-pH) could stably colonizationand reproduction in the cecum of chickens. It was also demonstrated that the recombinant Lactobacillus (DLD17-pH) not only could triggered the production of the specific anti-HAl IgA antibody level in the mucosa of respiratory and intestinal, but also could increase the anti-HAl IgG secreation in system level. Our research also proved that DLD17-pH could activate the cellular immunity and innate immune response to enhance the immune defense capability. Therefore, DLD17-pH could be a promising oral vaccine candidate against HPAI. The development of recombinant Lactobacillus (DLD17-pH) oral vaccine not only provides a new research idea for the prevention and control of the spread of avian flu, but also laid the foundation for the application of host lactobacillus as live vector delivery system. I will discuss it from the following the four aspects.
1. Isolation, identification and screening of lactic acid bacteria in chicken
Firstly,22Gram positive strain, harvested from the cecal mucosa of health adult chicken, were identified by amplified16s-rRNA fragments of each strain of bacteria, and compared with determining strains of species and strains.Combined with the results of biochemical fermentation strains, the five species of lactic acid bacteria strains in chicken were determine as the receptor into strains. In addition, five strains of acid tolerance of Lactobacillus from chicken, Caco-2cell adhesion, and antibiotic sensitivity were determined. To determine the optimal shock transformation of lactic acid bacteria transformation program, lactococcus vector plasmid plem415blank pmg36e and Lactobacillus vector plasmid transfer lactococcus1363and4356as Lactobacillus acidophilus transformation receptor positive control strain of electric shock, competent preparation and electron conditions of trial and error. After the optimal conversion options, the five strains of Lactobacillus from chicken to do transformation filter test of electric shock, the filter can accept of exogenous plasmid of Lactobacillus from chicken. These results suggestion that isolate and filter down to five strains of strain, a milk Neisseria gonorrhoeae strains of fecal gut DLD-1, a strain of Lactobacillus fermentation (DLD-5), three for salivary Lactobacillus DLD-9, DLD-13, DLD-17, respectively. Through electric shocks into screening test, we get a strain of Lactobacillus from chicken suitable for exogenous plasmid, the salivary Lactobacillus DLD-17.
2. Construction of recombinant H5N1haemagglutinin expression of lactic acid bacteria and its verification
In this chapter, we firstly designed two primer HAF1and HAF2containing restrictive enzyme SalⅠ and HindⅢ. The part of fragment HA1in H5N1Asia type high pathogenic sexual avian influenza (AIV) blood coagulation were cloned into pmd19-T vector. After measuring sequence validation,. Linearized vector pmg36e and purpose fragment HA1with restrictive enzyme SalⅠ and HindⅢ were transfer recombinant lactic acid milk Neisseria gonorrhoeae, named1363-EH, through electric shock transformation. SDS-page and Western blot results indicate that blood coagulation pigment protein in lactic acid milk Neisseria gonorrhoeae were correctly express. As the above, we also constructed the expression plasmid of recombinant hemagglutinin Lactobacillus pLEM415-pH. To clone the promoter p of lactic acid dehydrogenation enzyme (LDH), two primer P1and P2were designed. Then promoter p and blood coagulation pigment gene HA1were connected to Bacillus vector pLEM415, constructing of recombinant lactic acid Bacillus expression pLEM415-pH. Secondly, vector pLEM415-pH were transfer through the method of transformation of electric shock, obtained two recombinant hemagglutinin gene of strains of Lactobacillus, namely4356-pH and DLD17-pH. Protein electrophoresis and Western-blot were shown that the haemagglutinin of HA1were expressed in two recombinant Lactobacillus strains. In short, we have three strains of lactic acid bacteria for recombinant expression of hemagglutinin of avian influenza, namely restructuring lactococcus1363-EH, recombinant of Lactobacillus acidophilus4356-pH and DLD17-pH, which laid a solid foundation for following expression of hemagglutinin of avian influenza research of recombinant Lactobacillus oral vaccine.
3. Immunity levels of mouse after immunized orally with three recombinant lactic acid bacteria expressing H5
Observe antibody changes of mouse respiratory and gastrointestinal tract and whole body after oral immunized with three recombinant lactic acid bacteria1363-EH, DLD7-pH and LA4356-pH. Isolate spleen lymphocytes of immunized mouse and analyze the ratio ofCD4+/CD8+, CD19+lymphocyte subpopulations. Observe proliferation and secretion level of IFN-y and IL-4lymphocytes in vitro under the stimulus of specific antigen HAL Results indicated that IgA level was significantly increased in gastrointestinal tract after oral immunizing with this three bacteria; IgA level oral immunized with LL36EH and4356-pH was decreased with the lengthening of immunized time, while IgA level of mouse immunized with DLD-pH keep stability. These show that these three bacteria can induce local mucosal immune responses. In trachea, only chicken Lactobacillus DLD17-pH group show significant specific IgA secretion, a week later than the gut. We found that IgG antibody level and HI titers in the serum mice immunes with the three recombinant lactic acid bacteria are low, immunized, it is up to4.5(the Log2) of mice immune with specific DLD17-pH. Integrated with lymphocytes proliferation test and the ratio of CD4+/CD8+,CD19+lymphocyte subpopulations, we found that recombinant lactic acid bacteria can activate T lymphocytes and B lymphocytes in the body; which is mainly caused by the proliferation of Th2type the helper T cells, whereas no significant differences between the three bacteria. To some extent, three recombinant lactic acid bacteria which can express the avian influenza hem agglutinin can stimulate specifically humeral and cellular immune response, but immune effect different because of the strain source, characteristics. DLD17-pH did best in inducing humeral and cellular immune response, particularly it can effectively induce intestinal secretion of specific IgA antibodies in the respiratory mucosa. Herein, DLD17-pH is most suitable for mucosal immunity applied to the bird flu in poultry.
4. Study on Immune effect of recombinant chicken Lactobacillus DLD17-pH in chicks immunized orally
Immune chicks orally with restructured chicken Lactobacillus DLD17-pH to research local and systemic antibody level changes in respiratory and digestive tract in chicks. Detect the colonization of DLD17-pH in chick intestinal and compare immune effects with avian influenza inactivated vaccine. The results showed that DLD17-pH can not induce the production of specific IgA in the jejunum, cecal mucosa local and trachea, but also can induce specific low-level IgG antibodies. Compared with the subcutaneous immunization of avian influenza inactivated vaccine group, DLD17-pH can significantly increase gene expression of cytokines IFN-y in the digestive and respiratory tract organizations locally, TLR-2, and AvBD, While decrease gene expression of cytokines IL-4. Increasing of DLD17-pH in the cecum shows DLD17-pH can colonize in the cecal mucosa. It keeps relatively stable on21th day. In summary, oral administration of recombinant Lactobacillus DLD17-pH can not only activate early cytokine secretion in the digestive tract local tissue, respiratory, but also can induce specific antibodies of the avian flu in whole body. Therefore, it provides a new train of thought for prevents poultry bird flu and immunity by oral immunization recombinant Lactobacillus DLD17-pH.
引文
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