Immunological Adjuvant Function of Aluminium Phosphate and Chicken IL-18 in NDV F Gene Vaccine

[Objective] This study aimed to investigate the immunological adjuvant function of aluminium phosphate and chicken IL-18 in NDV F gene vaccine. [Method] The vaccine （0.2 ml） containing aluminum phosphate adjuvant （90 μg）, pcDNA/F （200μg）, and pcDNA/chlL-18 （200 μg） was prepared. The 7 d old chick- ens to be tested were randomly divided into six groups （12 chickens in each group） and immunized through intramuscular injection with inactivated Newcastle disease vaccines, pcDNA/F＋pcDNA/chlL-18＋phosphate aluminum, pcDNA/F, pcDNA/F.＋pcDNA/ chlL-18, pcDNA/F＋aluminum phosphate, and physiological saline respectively; the secondary immunization was conducted with the same dose when the chickens were 21 d old. Their blood was sampled 0, 7, 14, 21, 28 d after first immunization. Anti- body titer was detected with ELISA and T cell transformation rate was measured with MIT. Experimental chicken will be challenged with 30 LD50 NDV virulence 28 d after first immunization. [Result] The survival rate of the chickens immunized with pcDNA/F＋aluminium phosphate＋pcDNA/chlL-18 achieved 8/12, higher than that of those immunized with pcDNA/F 4/12 and pcDNA/F＋pcDNA/chlL-18 （6/12）. The NDV antibody titer of the chickens immunized with pcDNA/F＋ aluminum phosphate, pcD- NA/F＋pcDNA/chlL-18 and pcDNA/F＋pcDNA/chlL-18＋aluminum phosphate is not differ- ent （P〉0.05）, but significantly lower than that of the chickens immunized with tradi- tional vaccine （P〈0.05）. The T cell transformation rate of the chickens immunized with pcDNA/F＋pcDNA/chlL-18＋aluminium phosphate was obviously higher than that of the chickens immunized with pcDNA/F （P〈0.05）. The T cell transformation rates of chickens immunized with pcDNA/F and the traditional vaccine showed no signifi- cant difference （P〉0.05）. [Conclusion] Combination of aluminium phosphate and pcD- NA/chlL-18 can significantly enhance the immune effect of NDV F gene vaccine.

THE BIOLOGICAL CHARACTERISTICS OF ADENOVIRUS-MEDIATED IL-18 GENE-MODIFIED MURINE COLORECTAL ADENOCARCINOMA CELL IN VIVO AND IN VITRO

Objective: Interleukin 18 (IL-18) is a strong activator of NK cells and promotes the generation of IL-2, IFN-γ and GM-CSF. In the present study, we constructed adenovirus encoding IL-18 gene (AdIL-18), and observed the biological characteristics of IL-18 gene-modified murine colorectal adenocarcinoma cell (CT26)in vivo andin vitro. Methods: Gene modification was mediated by adenovirus. The proliferation of the cells was determined by MTT and IL-18 was assayed by ELISA. The cytotoxicity of NK and CTL was detected by four-hour51Cr release assay. Results: IL-18 gene modification had no effect on the proliferation and morphology of CT-26 cellsin vitro, but the growth of IL-18-modified CT26 cells was obviously inhibitedin vivo. In addition, although IL-18-modified CT26 cells could form tumor nodulesin vivo as well as LacZ-modified CT26 cells or wild-type CT26 cells, the mean survival time of the mice inoculated with IL-18-modified CT26 cells was significantly prolonged as compared with that of control groups. Thus, the anti-tumor immune responses were induced in the group of mice inoculated with IL-18-modified CT26 cells, which might be related to the activation of NK cells and CTL. However, all the three groups ultimately died of tumor. Conclusion: IL-18-modified CT26 cells could induce the anti-tumor immune responses incompletely, which required other factors for effective anti-tumor responses.

鸭IL-18基因克隆与序列分析

[目的]为深入研究鸭IL-18的基因表达及其生物学活性和分子佐剂的应用奠定基础。[方法]依据GenBank中香港鸭IL-18基因的序列设计1对特异引物,以广东水鸭脾淋巴细胞总RNA为模板进行RT-PCR扩增,克隆广东水鸭IL-18基因并进行序列分析。[结果]测序结果表明,广东水鸭的IL-18基因开放阅读框为603 bp,编码201个氨基酸,分子量为23.2 kDa。序列分析表明,所获得的广东水鸭IL-18基因与GenBank中香港鸭的核苷酸同源性为98.2%,氨基酸同源性为100%。它与鸡和火鸡的核苷酸同源性分别为86.9%和87.1%,而氨基酸同源性分别为96.5%和97.0%。[结论]进化树分析结果表明,鸭IL-18基因与鸡和火鸡进化关系较近。

Synthesis of Codon-optimized Human Interleukin-18 Gene by Combination of Chemical and Enzymatic Method

According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleotides, DNA fragments were designed and synthesized by the phosphoramidite four-step chemical method. The whole DNA sequence was synthesized by a one-step total gene synthesis method, and then inserted in pUC18 vector. Five positive clones identified by blue-white colony screening were sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The sequencing result shows that one clone contained the complete correct gene in all the five positive clones.

固始鸭白细胞介素-18全基因克隆与分子进化分析

根据GenBank发表的鸭IL-18cDNA基因序列设计、合成一对引物,应用RT-PCR技术,无需用非特异性免疫原如PHA等刺激脾淋巴细胞,直接提取脾淋巴细胞总RNA,扩增固始鸭IL-18基因,并克隆、测序。测序结果表明固始鸭IL-18基因全序列为610bp,包含1个完整阅读框,编码1条由200个氨基酸残基组成的多肽。序列分析发现,固始鸭IL-18基因与GenBank中两条鸭IL-18基因(AF336122和DQ490137)核苷酸同源性分别为98.8%、99.8%,与AF336122有5个碱基发生非同义变异,2个碱基为同义变异,氨基酸同源性为97.5%,与DQ490137有一个碱基发生非同义变异,氨基酸同源性为99.5%。固始鸭IL-18前体蛋白第30位谷氨酸处有一个IL-1β转换酶的caspase-1切割位点及在人和其他动物中已证实的IL-1标签序列,推测鸭IL-18成熟蛋白由170个氨基酸组成。固始鸭与人和其他动物的IL-18基因进化分析表明,IL-18基因存在着种的多样性,且亲缘关系越近,同源性越高。

樱桃谷鸭白介素18基因的克隆与原核表达

从鸡新城疫病毒刺激的鸭脾脏淋巴细胞培养液中提取基因组RNA,采用RT-PCR方法扩增鸭IL-18基因,插入pMD18-T载体,进行序列测定与分析。将成熟鸭IL-18基因亚克隆到表达载体PBCX中,并转化入大肠杆菌E.coliBL21进行表达。测序结果表明,该基因由603个核苷酸组成,包含一个完整阅读框,共编码201个氨基酸。表达载体经IPTG诱导表达出相对分子量约为59KD的融合蛋白。

Analysis Tissue Expression of IFN-γin IL-12 and/or IL-18 Gene Ablated Na(?)ve Mice

Interleukin 12(IL-12)and/or interleukin 18(IL-18)gene ablated mice were applied for the investigation of the tissue expression of interferon γ(IFN-γ).For IL-12^(-/-),IL-18^(-/-),IL-12^(-/-)/18^(-/-) and wt mice,reproductive performance were recorded and IFN-γ concentrations in heart,lung,liver,spleen,kidney and serum were quantified by ELISA. There were no significant differences of IFN-γ in heart,lung and kidney between 4 strains although control group was higher.It was observed that for IL-12^(-/-) mice,compared with other 3 groups,IFN-γ in liver and spleen were decreased(p<0.05)and reproductive performance appeared to be impaired.Serum IFN-γ level of IL-12^(-/-)/18^(-/-) mice was significantly higher(p<0.05).It was showed that IFN-γ productions under the normal condition were independent upon IL-12 and IL-18,its expressions in various tissues were different,and optimal IFN-γ is necessary for the normal growth and development of mammals.This study is helpful for clinical cytokines therapy.Cellular & Molecular Immunology.2005;2(1):68-72.

鸭源蛔虫分子生物学鉴定及临床病理分析

本试验通过对送检的1例病死番鸭进行剖检,在其肠道发现多量蛔虫;使用苏木精-伊红(H.E.)染色方法对病鸭肠道进行组织病理学分析,同时提取分离的3条蛔虫DNA,在传统形态学观察基础上,通过PCR技术进行18S rRNA基因片段的扩增和测序比对。结果显示:蛔虫寄生造成鸭肠道黏膜大量损伤脱落,并伴有大量的嗜酸性细胞浸润,鸭源蛔虫形态特征与鸡蛔虫相近,18S rRNA基因序列与鸡蛔虫(EF180058.1)的同源性高达100%,表明此次分离的3条鸭源蛔虫属于鸡蛔虫。综上,本试验对鸭源蛔虫进行分子生物学鉴定,为鸭蛔虫病的防控提供了相关参考。