输入性卵形疟原虫细胞色素b、细胞色素c氧化酶Ⅰ和乳酸脱氢酶基因单核苷酸多态性分析

摘要/Abstract

摘要：

目的对卵形疟原虫（Plasmodium ovale）curtisi亚种和wallikeri亚种编码的细胞色素b（cytochrome b,Cytb）,细胞色素c氧化酶Ⅰ（cytochrome c oxidaseⅠ,CoxⅠ）和乳酸脱氢酶（lactate dehydrogenase,LDH）基因的单核苷酸多态性进行分析。方法选择浙江省疟疾诊断参比实验室2011-2014年确诊的卵形疟原虫感染患者血样,提取卵形疟原虫DNA,巢式PCR扩增卵形疟原虫Cytb、CoxⅠ和LDH基因的部分片段,对扩增产物进行双向测序。测序序列经校对、拼接后,应用MEGA软件对测序序列和相应的氨基酸序列进行比对,分析单核苷酸多态性位点以及氨基酸突变位点。结果 19例卵形疟原虫感染者（其中curtisi亚种11例,wallikeri亚种8例）均为赴非洲返国人员。PCR扩增Cytb、CoxⅠ和LDH基因的产物长度分别为735、1 323和355 bp。curtisi和wallikeri两个亚种在Cytb、CoxⅠ和LDH基因分别发现10、13、13个单核苷酸二态性位点,且大多数的二态性位点属于同义突变。此外,两个亚种内部也存在核苷酸多态性,同时发现1例血样的CoxⅠ基因存在693个碱基的缺失。结论对卵形疟患者血样的Cytb、CoxⅠ和LDH基因进行了单核苷酸多态性分析,丰富了卵形疟原虫基因多态性信息。

关键词: 卵形疟原虫, 单核苷酸多态性, 巢式PCR

Abstract:

Objective To analyze the single nucleotide polymorphism (SNP) of cytochrome b (Cytb), cytochrome c oxidaseⅠ (CoxⅠ) and lactate dehydrogenase (LDH) genes of Plasmodium ovale curtisi and P. ovale wallikeri. Methods The P. ovale samples identified in Zhejiang Provincial Reference Laboratory for Malaria Diagnosis during 2011-2014 were used for nested PCR to amplify the partial fragments of Cytb, CoxⅠ and LDH genes. The amplified products were sequenced in both directions. The nucleotide sequences and their corresponding amino acid sequences were aligned and analyzed using the Mega software to find out the SNP sites and amino acid mutation sites. Results The 19 samples of P. ovale (11 curtisi and 8 wallikeri) were all from persons returning from Africa. The lengths of PCR products for Cytb, CoxⅠ and LDH were 735, 1 323 and 355 bp, respectively. There were 10, 13 and 13 single nucleotide dimorphism sites in Cytb, CoxⅠ and LDH, respectively, and most were synonymous mutation. Meanwhile, some nucleotide polymorphisms were found between the two subspecies. The CoxⅠ gene was found to harbor a 693 bp deletion mutation in one sample. Conclusion We performed SNP analysis on Cytb, CoxⅠ and LDH genes of P. ovale, which will enrich the knowledge on gene polymorphism of P. ovale.

Key words: Plasmodium ovale, Single nucleotide polymorphism, Nested PCR

中图分类号:

R382.31

张玲玲, 姚立农, 陈华良, 陆巧绎, 阮卫. 输入性卵形疟原虫细胞色素b、细胞色素c氧化酶Ⅰ和乳酸脱氢酶基因单核苷酸多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(5): 429-433.

Ling-ling ZHANG, Li-nong YAO, Hua-liang CHEN, Qiao-yi LU, Wei RUAN. Single nucleotide polymorphism analysis of cytochrome b, cytochrome c oxidaseⅠand lactate dehydrogenase genes of imported Plasmodium ovale[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(5): 429-433.

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参考文献 17

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引物名称 Primer name	引物序列（5′→3′） Primer sequence（5′→3′）	基因 Gene	退火温度 Annealing temperature
PLAS1	GAGAATTATGGAGTGGA- TGGTG	Cytb	第一轮： 55 ℃
PLAS2a	GTGGTAATTGACATCCW- ATCC		First circle: 55 ℃
PLAS3	GGTGTTTYAGATAYATGCAYGC		第二轮： 55 ℃
PLAS4	CATCCWATCCATARTA-WAGCATAG		Second circle: 55 ℃
COX 1a	CGCCTGACATGGATGGA-TAATAC	CoxⅠ	第一轮： 53 ℃
COX 1b	CCATTTAAAGCGTCTGG-ATAATC		First circle: 53 ℃
COX 1c	GATTAACCGCTGTCGC-TGGGACTG		第二轮： 58 ℃
COX 1d	CGTCTAGGCATTACATT-AAATCC		Second circle: 58 ℃
LDH1	GGNTCDGGHATGATHGGAGG	LDH	第一轮： 55 ℃
LDH2	GCCATTTCRATRATDGC-AGC		First circle: 55 ℃
LDH7	TGTDATGGCWTAYTCVA-ATTGYMARGT		第二轮： 52 ℃
LDH8	CCATYTTRTTNCCATGW-GCWSCDACA		Second circle: 52 ℃

病例 No. case	性别 Sex	年龄Age	年份Year	感染来源国Source country	镜检结果Microscopy	PCR结果PCR results
1	男Male	33	2011	尼日利亚Nigeria	-	Poc
2	女Female	34	2012	利比里亚Liberia	Po	Poc
3	女Female	31	2012	利比里亚Liberia	Po	Poc
4	男Male	25	2012	加纳Ghana	Pv	Poc
5	男Male	38	2012	加纳Ghana	Pv	Pow
6	男Male	32	2012	刚果布Congo	Po	Poc
7	男Male	25	2012	赤道几内亚Equatorial Guinea	Po	Poc
8	男Male	37	2012	利比里亚Liberia	Pm	Pow
9	男Male	41	2012	加纳Ghana	Po/Pf	Pow
10	男Male	40	2013	加纳Ghana	Po	Poc
11	男Male	55	2013	尼日利亚Nigeria	Po	Pow
12	男Male	30	2013	尼日利亚Nigeria	Po	Pow
13	男Male	21	2013	加纳Ghana	Po/Pv	Poc
14	男Male	44	2013	加纳Ghana	Pv	Poc
15	男Male	47	2014	尼日利亚Nigeria	Po	Pow
16	女Female	35	2014	赤道几内亚Equatorial Guinea	Pv	Poc
17	男Male	25	2014	尼日利亚Nigeria	Pv	Poc
18	男Male	34	2014	喀麦隆Cameroon	Po	Pow
19	女Female	33	2014	南非South Africa	Pv	Pow

种名Species name	472	499	589	631	841	853	871	889	982	1000	1045
卵形疟原虫curtisi亚种 P. ovale curtisi	T	C	T	C	G	C	T	T/C	C	C	T
卵形疟原虫walliker亚种 P. ovale wallikeri	A	T	A	T	T	T	A	T	T	T	A
核苷酸二态性位点 Amino acid substitution	-	-	-	-	M248I	-	-		-	-	-

种名Species name	3390	3398	3590	3599	3603	3614	3716	3773	3798	3971	4107	4223	4254	4496
卵形疟原虫curtisi亚种 P. ovale curtisi	C	C	T	T	C	C	T	G	C	C	A	T	A	A
卵形疟原虫walliker亚种 P. ovale wallikeri	T	A	C	C	T	T	G	A	T	T	G	A	A/C	T
核苷酸二态性位点 Amino acid substitution	-		-	-	-	-	-	M176I	-	-	I288V	-	N337H	-

种名Species name	175	217	223	238	281	301	319	386	407	455	484	487	490
卵形疟原虫curtisi亚种 P. ovale curtisi	C	T	C	T	T	T	A	T	T	C	T	T	T
卵形疟原虫walliker亚种 P. ovale wallikeri	T	A	T	A	C	C	T	C	C	T	G	A	C
核苷酸二态性位点 Amino acid substitution	-	-	-	-	-	-	-	-	S136P		N161K	-	-