本文選題：Sabin株　切入點：脊髓灰質炎病毒滅活疫苗　出處：《中國協和醫(yī)科大學》2009年碩士論文

【摘要】： 隨著全球消滅脊髓灰質炎(脊灰)目標的臨近,疫苗衍生病毒(VDPV)的流行、疫苗相關麻痹型病例(VAPP)的發(fā)生等對無脊灰狀態(tài)成果的鞏固威脅很大。在消滅脊灰的最后階段,應盡快消滅脊灰野毒株,并使用滅活疫苗(Inactivatedpoliovirus vaccine,IPV)替代目前廣泛使用的脊髓灰質炎減毒活疫苗(Oral poliovirusvaccine,OPV)來維持無脊灰狀態(tài),消除自然界的活病毒。對于新的生產廠家考慮到疫苗生產的生物安全性等因素,WHO鼓勵研制Sabin株脊髓灰質炎滅活疫苗(Sabin IPV)。 目前尚無成品Sabin IPV上市,于是Sabin IPV的定量檢測、質量指標和免疫劑量等都還沒有統(tǒng)一標準。前期研究顯示,Sabin IPV與野毒株IPV(WT-IPV)在抗原性、穩(wěn)定性等方面均有差別,于是建立在WT-IPV基礎上的定量檢測方法和質量控制指標并不適用于Sabin IPV。另外,Sabin IPV與WT-IPV抗原性也有差異,Sabin2型的免疫原性只是野毒株MEF-1株的1/10。為解決疫苗免疫原性不佳的問題,最常用的方法即添加疫苗佐劑。本論文旨在于建立適用于Sabin IPV的D抗原定量方法及蛋白質含量測定方法,完善Sabin IPV檢定和質量控制體系;并進一步探索SabinIPV中添加佐劑對免疫效果產生的影響,探討Sabin IPV中佐劑應用的意義和最適劑量等。 本研究第一部分即建立Sabin株脊髓灰質炎病毒滅活疫苗(Sabin IPV)中D抗原含量及蛋白質含量的檢測方法。首先通過摸索各步驟反應條件,優(yōu)化本實驗室建立的多抗ELISA法測定Sabin IPV中D抗原含量的體系,對3個批次的樣品進行5次重復測定,結果的變異系數均小于10%。另外,利用蛋白質遇三氯乙酸產生沉淀的特點,對Lowry法進行改良,建立了能夠準確測定Sabin IPV中微量蛋白質含量的方法,實驗證明,該方法能夠排除Sabin IPV中的游離氨基酸、多肽和酚紅指示劑等物質的干擾,線性范圍為2.5～40ug/ml,r=0.9998,最佳條件下加標平均回收率為95.32%,精密度試驗結果批內和批間變異系數均小于10%。 研究的第二部分通過將Al(OH)_3、DC-Chol和Al(OH)_3+DC-Chol 3種不同疫苗佐劑按不同劑量與Sabin IPV(每劑含D抗原Ⅰ型15DU、Ⅱ型16 DU和Ⅲ型22.5 DU)配伍后分別免疫Wistar大鼠,主要檢測各組大鼠免疫后血清中和抗體水平、特異性IFN-γ的產生情況以及免疫后各組大鼠CD3/CD4/CD8分子表達的差異。綜合上述結果評價在Sabin IPV中添加Al(OH)_3或/和DC-Chol對大鼠體液免疫和細胞免疫的影響。通過微量中和實驗檢測Sabin株脊髓灰質炎特異性中和抗體發(fā)現,在Sabin IPV中添加佐劑,普遍于第1針免疫后提高了大鼠血清中3個型特異性中和抗體陽轉率;而且3種佐劑還在第2針免疫后提高了Ⅱ型中和抗體的陽轉率;Sabin IPV中添加佐劑后,對特異性中和抗體的水平也有不同程度的影響。添加Al(OH)_3或DC-Chol在第2針免疫后就產生了高效價的Ⅰ型中和抗體,如添加0.5mgDC-Chol第2針免疫后Ⅰ型中和抗體效價幾何均數(GMT)達到未添加佐劑組的3倍以上,顯著提高了早期Ⅰ型中和抗體的水平;添加0.1mgAl(OH)_3組與未添加佐劑組相比,第3針免疫后Ⅱ型特異性中和抗體效價的幾何均數增長2倍以上;添加Al(OH)_3或DC-Chol或Al(OH)_3+DC-Chol聯合佐劑在第2針免疫后就獲得了高水平的Ⅲ型特異性中和抗體,GMT比未添加佐劑組增長15倍和12倍,即大大提高了早期Ⅲ型中和抗體的水平。ELISPOT測定針對Sabin株脊髓灰質炎病毒的特異性IFN-γ的結果顯示,注射不含佐劑Sabin IPV的大鼠未產生特異性IFN-γ斑點,注射添加了Al(OH)_3或DC-Chol佐劑Sabin IPV的大鼠產生了少量特異性IFN-γ斑點,其中添加0.25mgDC-Chol組效果最好,Ⅰ、Ⅱ、Ⅲ型各產生特異性IFN-γ斑點22.25±6.24、13.75±3.77和22.50±5.92 SFC/10~6cell提示添加Al(OH)_3或DC-Chol佐劑可能對大鼠針對Sabin株脊髓灰質炎病毒的特異性細胞免疫有少許增強作用;流式細胞儀檢測各組大鼠CD3/CD4/CD8分子表達的差異的結果顯示,Al(OH)_3或DC-Chol佐劑的應用能提高大鼠CD3~+CD4~+/CD3~+CD8~+值,提示2種佐劑的應用均能增強大鼠的免疫功能,但這種增強作用在佐劑的不同劑量組之間差別不顯著。綜上所述,Al(OH)_3或DC-Chol能夠增強Sabin IPV的體液免疫效果,對細胞免疫也有一定增強作用,能在更短的時間內使Wistar大鼠獲得更好的保護效果。
[Abstract]:With the global eradication of poliomyelitis (polio) target approaching, vaccine derived virus (VDPV) epidemic, the vaccine associated paralytic (VAPP) occurrence of polio free efforts to consolidate the threat. In the final stage of polio eradication, should as soon as possible to eradicate poliomyelitis wild strains, and the use of inactivated vaccine (Inactivatedpoliovirus vaccine IPV) to replace the widely used live attenuated poliomyelitis vaccine (Oral, poliovirusvaccine, OPV) to maintain polio free, eliminate the live virus in nature. For new products taking into account biological safety factors such as vaccine production, WHO encourages the development of Sabin inactivated poliovirus vaccine (Sabin IPV).
At present there is no finished Sabin IPV listed, so the quantitative detection of Sabin IPV, quality index and immune dose so there is no uniform standard. Previous studies have shown that Sabin IPV IPV (WT-IPV) and wild strains in antigenicity, stability and other aspects were different, and based on WT-IPV on the basis of the quantitative detection method and the quality control index is not applicable to Sabin Sabin IPV and WT-IPV IPV. in addition, there are differences in immune antigenicity, Sabin2 type of just wild strain MEF-1 1/10. to solve the problem of poor immunogenicity of vaccine, the most common method is to add adjuvant vaccine. The purpose of this paper is to establish methods for the quantitative determination method of D antigen for Sabin IPV Sabin IPV and protein content, improve the verification and quality control system; and to further explore the effect of adjuvant SabinIPV on immune effect of adjuvant Sabin, IPV should be used and the significance The optimum dosage.
The first part of this study is to establish the Sabin strain of inactivated poliovirus vaccine (Sabin IPV) method for detection of antigen content and protein content in D. First, through exploring the various steps of the reaction conditions, the content of Sabin IPV in D antigen determination optimization polyclonal antibody ELISA method established in our laboratory system of 3 batches of samples were repeated 5 times determination of the coefficient of variation of the precipitation was less than 10%. in addition, based on the characteristics of protein with three chloroacetic acid, modified Lowry method, established a method for accurate determination of trace protein content of Sabin in IPV, the experiment shows that this method can eliminate the free amino acid Sabin IPV, peptides and other substances interfere with phenol red indicator the linear range is 2.5 ~ 40ug/ml, r=0.9998, under the optimum conditions the average recovery rate was 95.32%, the results of precision test intra and interassay coefficients of variation were less than 10%.
The second part of the study by Al (OH) _3, DC-Chol and Al (OH) _3+DC-Chol 3 different adjuvants according to different dosage of Sabin and IPV (each agent containing D antigen of 15DU type I, II and III 16 DU 22.5 DU) and after Wistar rats were immunized, mainly detected in rats with immune the serum levels of neutralizing antibodies, specific IFN- gamma production and expression of rats after CD3/CD4/CD8 molecular and immunological differences. The results of evaluation in Sabin IPV add Al (OH) effect of _3 or / and DC-Chol on humoral immunity and cellular immunity in rats. By micro neutralization assay of Sabin poliovirus specific neutralizing antibody found that adding adjuvant in Sabin IPV, generally first needles after immunization increased 3 rat serum specific neutralizing antibody positive conversion rate was second; and 3 adjuvants can improve immune needle type II neutralizing antibody positive rate; Sabin IPV In addition to adjuvant, specific neutralizing antibody level has different effects. Adding Al (OH) type _3 or DC-Chol produced high titer in second needle immune neutralizing antibodies after adding 0.5mgDC-Chol second pin type 1 immune neutralizing antibody titer and geometric mean (GMT) was added to the adjuvant group has more than 3 times, significantly improve the early type of neutralizing antibody level; adding 0.1mgAl (OH) _3 group compared with the group without adjuvant, geometric neutralizing antibody titer after immunization third needle type II specific mean growth of more than 2 times; adding Al (OH) _3 (OH or DC-Chol or Al _3+DC-Chol) combined with adjuvant have high levels of type III specific neutralizing antibody in second needle immunized GMT than without adjuvant group increased 15 times and 12 times, which greatly increased the level of.ELISPOT in the early stage of type III Determination of neutralizing antibody specific for IFN- Sabin strains of polio virus Gamma results showed that injected without adjuvant Sabin IPV rats did not produce specific IFN- gamma spots, injection with Al (OH) _3 or DC-Chol Sabin IPV adjuvant in rats produced a few specific IFN- gamma spots, one of the best, adding 0.25mgDC-Chol group I, II, III type specific IFN- dot gamma 22.25 + 6.24,13.75 + 3.77 and 22.50 + 5.92 SFC/10~6cell Al (OH) suggested that adding _3 or DC-Chol adjuvant may have a little effect to enhance specific cellular immunity against poliomyelitis virus Sabin strain rats; the differential expression of /CD8 molecule CD3/CD4 in rats were determined by flow cytometry showed that Al (OH) the application of _3 or DC-Chol adjuvant can improve rat CD3~+CD4~+/CD3~+CD8~+, suggesting that the 2 Application of adjuvant can enhance the immune function of rats, but this enhancement between different adjuvant dose group was no significant difference. In summary, Al (OH) _ 3 or DC-Chol can enhance the humoral immunity effect of Sabin IPV, and enhance cell immunity. It can make Wistar rats get better protective effect in a shorter time.

【學位授予單位】：中國協和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

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